Characterization of the ompL1 gene of pathogenic Leptospira species in China and cross-immunogenicity of the OmpL1 protein

Background The usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp. Identification of genus-specific protein antigens (GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1, a transmembrane porin of pathogenic leptospires, was identified as a possible GP-Ag, but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown. Results PCR analysis demonstrated that the ompL1 gene existed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated in China. In the standard strains, the ompL1 gene could be divided into three groups (ompL1/1, ompL1/2 and ompL1/3) according to their sequence identities. Immune electron microscopy demonstrated that all products of the different gene types of ompL1 are located on the surface of leptospires. The microscopic agglutination test revealed extensive yet distinct cross-immunoagglutination among the antisera against recombinant OmpL1 (rOmpL1) and leptospiral strains belonging to different ompL1 gene types. These cross-immunoreactions were further verified by ELISAs using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients. All the antisera against rOmpL1 proteins could inhibit L. interrogans strain Lai from adhering to J774A.1 cells. Furthermore, immunization of guinea pigs with each of the rOmpL1 proteins could cause cross-immunoprotection against lethal challenge with leptospires from different ompL1 gene types. Conclusion Three types of the ompL1 gene are present in pathogenic leptospires in China. OmpL1 is an immunoprotective GP-Ag which should be considered in the design of new universal vaccines and serodiagnostic methods against leptospirosis.


Background
Leptospirosis, caused by infection with pathogenic Leptospira species belonging to different serogroups and serovars, is one of the most prevalent zoonotic diseases in the world [1][2][3]. A wide variety of serogroups and serovars have been identified along with endemicity which varies from region to region [4][5][6]. The leptospiral vaccines used currently are mainly multivalent dead whole-cell mixtures made of several local dominant serovars in different countries and regions. These vaccines, however, do not confer cross-protective immunity to the serogroups that are not represented in the vaccine [7,8], allowing the unrepresented serovars to continue causing outbreaks of leptospirosis. For instance, in a central province, Anhui, and an eastern province, Zhejiang, of China, L. interrogans serogroup Sejroe serovar Medanensis caused local outbreaks of leptospirosis [9][10][11][12]. In addition, vaccination with the whole-cell vaccines may lead to incomplete, short-term immunity as well as serious side effects [13][14][15]. A universal vaccine against leptospirosis is not available yet, making the identification of genus-specific protein antigens (GP-Ag) that display extensive crossimmunity very valuable for developing new vaccines and serodiagnostic methods.
Outer membrane proteins (Omps) are important pathogenic components and highly conserved in different serogroups and serovars of pathogenic leptospires. OmpL1, a transmembrane Omp with 320 amino acid residues, first reported by Haake and his colleagues in 1993, is a porin expressed by all the tested pathogenic Leptospira species [16][17][18][19]. However, the diversity of ompL1 gene sequences from different pathogenic Leptospira spp. and the distribution of the ompL1 gene in clinical isolates had not been characterized until now. Moreover, the cross-immunogenicity and immunoprotective effects of OmpL1 were mostly unknown.
In this study, we sequenced and analyzed ompL1 genes cloned from standard pathogenic strains of leptospires prevalent in China. Several prokaryotic recombinant products of the gene (rOmpL1) were expressed and their rabbit antisera were prepared. rOmpL1-based ELISAs were established to examine specific antibodies in sera from leptospirosis patients. In parallel, the microscopic agglutination test (MAT) was performed to detect the crossimmunoagglutination of different antisera against rOmpL1 proteins, and immunoelectron microscopy (IEM) was employed to localize OmpL1 on the leptospires. Finally, immunoprotection of rOmpL1 was tested in guinea pigs. Taken together, our results suggest that OmpL1 could be used as a major component of a universal and efficient immunogen for vaccination and also for diagnosis of leptospirosis.

Amplification of ompL1 genes in the standard and clinical strains
All the 15 standard strains and 163 isolates of pathogenic Leptospira species (Table 1) carried the ompL1 gene, since amplicons with the expected size were produced by PCR from all the strains and isolates (data no shown).

Molecular phylogeny and gene-typing of ompL1 gene
Based on the molecular phylogenetic relationship of their nucleotide sequences (GenBank Accession No.: AY622658-AY622672) and putative amino acid sequences ( Figure 1, see Additional file 1 for details), ompL1 genes from the pathogenic leptospires could be classified into three groups: ompL1/1, ompL1/2 and ompL1/ 3 ( Table 2). Sequence identity of the putative amino acid sequences between ompL1/1 and ompL1/2, ompL1/1 and ompL1/3, and ompL1/2 and ompL1/3 gene types was 92.50%-93.44%, 85.31%-86.88%, and 85.31%-86.25%, respectively. In addition, the ompL1 gene sequencing data of 39 clinical strains also supported the classification into three groups (Table 2). Both the nucleotide and putative amino acid sequence identities among the clinical strains with the same ompL1 gene type were above 98% (data not shown).

Identification of expressed rOmpL1 proteins and titers of rabbit antisera
rOmpL1/1, rOmpL1/2 and rOmpL1/3 were well expressed after induction with IPTG and showed single bands in gel after purification by Ni-NTA-chromatography ( Figure 3). Western blot analysis also indicated that serum samples from leptospirosis patients could recognize rOmpL1 proteins (Figure 4), implying that the proteins are immunogenic during natural infection. Immunodiffusion titers of all the rabbit antisera against rOmpL1 proteins were at least 1:4.

Detection of antibodies against rOmpL1 proteins in leptospirosis patients
In ELISA assays using rOmpL1/1, rOmpL1/2 and rOmpL1/3, the positive rates for IgG in specimens from 385 leptospirosis patients using 1 Table 4).

Immunoprotection due to immunization with rOmpL1 proteins
Immunization of guinea pigs with rOmpL1/1, rOmpL1/2 and rOmpL1/3 conferred a significant level of resistance against lethal challenge with pathogenic leptospires from the three different ompL1 groups (Table 5). However, the immunoprotective rates against the same ompL1 gene type leptospiral infection as the immunizing recombinant protein (62.5%-87.5%) were higher than when strains and proteins from different ompL1 gene types were used (25.0%-62.5%).

Discussion
Outer membrane proteins exposed on the surface of leptospires are known to react with the host cell and environment. Interestingly, lipopolysaccharide fractions confer protective immunity against challenge with homologous but not heterogonous leptospires, whereas protein extract induced significant protection against both types of challenge [20]. Thus far, a number of outer membrane proteins of leptospires, such as OmpL1, LipL32, LipL36, LipL41, LigA and LigB, have been cloned and characterized, and some of them have been shown to stimulate specific immunity in animal models [21][22][23][24]. Among all the leptospiral Omps, OmpL1 is a unique transmembrane protein that was confirmed to function as a porin, contribute to the survival of leptospires, and display synergetic immunoprotection with LipL41 [16][17][18]. However, major questions such as the distribution of ompL1 gene types in leptospiral strains, the exact localization of OmpL1, and cross-immunogenicity and immunoprotective effects of OmpL1 proteins remain unaddressed.
This study reveals that the ompL1 gene is present in the genomes of all the pathogenic leptospires tested. According to our alignment and phylogenetic analysis from the 15 standard strains of pathogenic Leptospira spp., three groups of ompL1 (ompL1/1, ompL1/2 and ompl/3) exist. However, the predicted secondary structure of the OmpL1 proteins revealed that there is little difference among the three groups. Thus, the differences in nucleotide sequences in the ompL1 gene types may not affect the immunogenicity and OmpL1 proteins, identifying OmpL1 as a genus-specific protein antigen.
Surface exposure is a key characteristic for an effective antigen. Although OmpL1 may be an outer membrane protein according to previous reports, the precise localization of OmpL1 still remained unclear. Leptospires possess both inner and outer membranes, but only the proteins expressed in the outer membrane are capable of interacting with the host immune system. To begin to characterize the localization, we used the prokaryotic recombinant expression technique to obtain a large amount of homogeneous OmpL1 proteins for preparation of immunoresponsive antisera from rabbits. Visualization by immunoelectron microscopy using anti-OmpL1 anti-sera confirmed that OmpL1 is located at the surface of the outer membrane of leptospires. MAT is a standard method for serodiagnosis of leptospirosis and serological classification of leptospires, for which live leptospiral cells are typ- ically used [23]. In this study, we used MAT to examine cross-immunoagglutination among the antisera from rOmpL1 proteins and a large number of strains belonging to different pathogenic leptospiral serogroups/serovars. The results indicated that there is extensive cross-immunoagglutination between the different ompL1 gene types of pathogenic leptospiral strains and the OmpL1 antisera, and not surprisingly, the highest agglutination was observed between antisera from the same ompL1 gene types as the leptospiral strains. Furthermore, ELISA results revealed that OmpL1s-specific antibodies are produced in all the sera from leptospirosis patients, and the trends in cross-immunoreactivities in the different ELISA tests are also similar to those of MAT.
At least 75 serovars of pathogenic leptospires belonging to 18 serogroups have been found to date in China, but only a few of them frequently cause leptospirosis. According to the annual reports on leptospirosis from Chinese CDCs, L. interrogans serogroup Icterohaemorrhagiae serovar Lai is the most dominant pathogenic leptospires, responsible for approximately 75% of the morbidity in the country. The other pathogenic leptospires causing leptospirosis are the Grippotyphosa, Autumnalis, Australis, Pomona and Hebdomadis serovars [3,11,25]. Our study showed that all the 163 clinical strains of different pathogenic Leptospiral serogroup/serovars belong to either the ompL1/1 or ompL1/2 group (data not shown). Thus, we conclude that leptospires from the ompL1/1 and ompL1/2 groups are the most prevalent strains in China.
In this study, immunization with either rOmpL1/1, rOmpL1/2 or rOmpL1/3 proteins could enhance significantly survival of guinea pigs lethally challenged with pathogenic Leptospira species (Table 5). The immunoprotective rates in the groups, in which animals received a rOmpL1 for immunization and challenge with the same ompL1 gene type, appeared to be higher than those being challenged with different ompL1 gene types.
In a previous study [35], we found L. interrogans serovar Lai strain Lai could adhere to macrophages, and we here show that adherence to macrophages could be blocked with antisera against any of the rOmpL1 proteins, confirming cross-immunogenicity of OmpL1 proteins from the three different groups. All these data collectively suggest that OmpL1 is likely a genus-specific antigen.

Conclusion
OmpL1 is a transmembrane protein extensively expressed in pathogenic leptospires. Although the ompL1 gene can belong to any of three different gene types, the OmpL1 proteins expressed by different gene types have conserved immunogenicity and the specific antibodies generally exist in sera of leptospirosis patients. OmpL1 should thus be viewed as a potential candidate of genus-specific antigen for the development of new universal vaccines and serodiagnostic methods for leptospirosis.  (Table 1). All the leptospiral strains were cultured in liquid Korthof medium containing 8% rabbit serum at 28°C [2,25]. All the patients were clinically diagnosed suffering from acute leptospirosis based on their medical history and clinical symptoms, including fevers, jaundice, hemorrhaging, myalgia and lymphadenectasis, as well as laboratory examination. Finally, 36 sera samples with negative results by MAT from healthy individuals for routine soma-Expression and purification of recombinant OmpL1 proteins Figure 3 Expression and purification of recombinant OmpL1 proteins. Lanes 1 and 6: protein markers (BioColor); lane 2: pET42a with no inserted ompL1 genes; lanes 3 to 5: the expressed rOmpL1/1, rOmpL1/2 and rOmpL1/3, respectively; lanes 7 to 9: the purified rOmpL1/1, rOmpL1/2 and rOmpL1/3, respectively.
toscopy were offered by the First Affiliated Hospital of Zhejiang University. Informed written consent for sample collection was obtained from all participants and individuals with ethical approval from the Ethics Committee of Zhejiang University. This research was conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health. All experimental protocols were approved by the Ethics Committee of Zhejiang University.

Cell line and cell culture
The murine monocyte/macrophage-like cell line, J774A.1, was provided by the Cell Bank of the Institute of Cell Biology in Shanghai, Chinese Academy of Science, and was maintained in RPMI 1640 medium (GiBco, Grand Island, The localization of OmpL1 on the surface of leptospires Figure 5 The localization of OmpL1 on the surface of leptospires. A: negative control; B to D: the immunogold particles binding to OmpL1/1, OmpL1/2 and OmpL1/3 on the surface of L. interrogans serovar Autumnalis strain Lin 4, serovar Lai strain Lai, and L. borgpetersenii serovars Ballum strain Pishu, respectively. USA) supplemented with 10% fetal bovine serum (FBS) (GiBco), and 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were cultured in 5% CO 2 at 37°C.

Amplification and sequencing of ompL1 genes
Leptospiral DNA was extracted using Bacterial Genomic DNA Extraction Kit (BioColor BioScience & Technology Co., Shanghai, China) and then dissolved in TE buffer. Density and purity of the extracted DNAs were detected by UV spectrophotometery. One pair of primers was applied to amplify the entire ompL1 gene. The upstream primer was 5'-CCG CATATG (Nde I) ATC CGT AAC ATA AGT AAG-3' and the downstream primer was 5'-CCG CTCGAG (Xho I) GAG TTC GTG TTT ATA ACC-3'. A High Fidelity PCR Kit (TaKaRa, Dalian, China), in which Taq-Pfu mixture is used as DNA polymerase, was used to amplify the target gene. The total volume per PCR was 50 μl which included 20 pmol of each of the primers, 2.5 U Taq-Pfu DNA polymerase, and 100 ng DNA templates. The reaction mixture was initiated by incubation at 94°C for 5 min, followed by 30 cycles of amplification at 94°C for 30 s, 55°C for 30 s and 72°C for 90 s, and then incubation at 72°C for 10 min. The products were detected in 1.5% ethidium bromide pre-stained agarose gel by agarose electrophoresis. The target products were predicted to be 960 base pairs (bp) in size. To obtain more accurate sequence data, the PCR products were purified using PCR Products Purification Kit (BioColor) and then ligated into plasmid pMD 18-T using T-A Cloning Kit (TaKaRa) [26]. The cloned ompL1 genes of 15 standard strains and 39 clinical strains were sequenced by Invitrogen (Shanghai, China).

Phylogenetic analysis and secondary structure prediction of OmpL1 protein
The similarity and homology of sequences were first evaluated with the BLASTN program of NCBI. Alignments were carried out using ClustalX 1.83 software [27] and manually adjusted using GeneDoc 2.7 software. The amino acid sequences were deduced from their nucleotide sequences. Phylogenetic analysis was carried out with Maximum Parsimony (MP) and Maximum Likelihood (ML) optimality criteria of the PAUP* package (Version 4.0b10) [28] and MEGA 3.1 software [29]. And the probability prediction for secondary structures [30,31], including antigenic analysis [32] of OmpL1 proteins, was computed using the protean program in DNAStar™ software package.

Prokaryotic expression and preparation of antisera against OmpL1 proteins
The recombinant plasmids pMD18-T-ompL1/1/2/3 as well as vector pET42a (Novagen, USA) were respectively On the 15th day after the last immunization, the rabbit sera were collected and the Immunodiffusion test was used to examine the titers of antisera [24].

Western blot assay
Western blot analysis was used to identify the immunoreactivity of rOmpL1 proteins using 12 serum samples from the patients diluted at 1:1000 (the same dilution of normal human sera was used as control) as the primary antibodies, and 1:3000 diluted HRP-labeled goat anti-human IgG (Jackson ImmunoResearch Laboratories Inc., USA) as the secondary antibody.

Microscopic agglutination test
The microscopic agglutination test (MAT) was used to determine cross-immunoagglutination under dark-field microscopy using rabbit antisera against the rOmpL1 proteins reacting with the 15 freshly-cultured standard strains Inhibition of leptospiral adherence to J744A.1 macrophages in the presence anti-rOmpL1 antiserum Figure 6 Inhibition of leptospiral adherence to J744A.1 macrophages in the presence anti-rOmpL1 antiserum. A: Leptospires binding to macrophages in the presence of irrelevant antisera. B: inhibition of leptospiral binding to macrophages in the presence of antiserum against recombinant OmpL1 protein (amplification ×1000).