Molecular identification of the ompL 1 gene within Leptospira interrogans standard serovars

Introduction: Leptospirosis, caused by infection with pathogenic Leptospira species, is one of the most prevalent zoonotic diseases in the world. Current leptospiral vaccines are mainly multivalent dead whole-cell mixtures made of several local dominant serovars. Therefore, design and construction of an efficient recombinant vaccine for leptospirosis control is very important. OmpL1 is an immunogenic porin protein that could be of special significance in vaccination and serodiagnosis for leptospirosis. Methodology: Three strains belonging to pathogenic L. interrogans were analyzed. The specific primers for proliferation of the ompL1 gene were designed. The amplified gene was cloned. In order to investigate the ompL1 nucleotide sequence and homological analysis of this gene, ompL1 genes cloned from standard vaccinal Leptospira serovars prevalent in Iran were sequenced and cloned. Results: PCR amplification of the ompL1 gene using the designed primers resulted in a 963 bp ompL1 gene product. The PCR based on the ompL1 gene detected all pathogenic reference serovars of Leptospira spp. tested. Based on alignment and phylogenetic analysis, although the ompL1 nucleotide sequence was slightly different within three vaccinal serovars (100%-85% identity), amino acid alignment of the OmpL1 proteins revealed that there would be inconsiderable difference among them. Conclusion: The ompL1 gene of the three isolates was well conserved, differing only by a total of 6 bp and the proteins by 2 amino acids. The cloned gene could be further used for expression and recombinant OmpL1 as an efficient and conserved antigen, and may be a useful vaccine candidate against leptospirosis in our region.


Introduction
Leptospirosis is one of the most important zoonoses with worldwide distribution.The disease occurs mostly in tropical, subtropical, temperate, and humid regions with high rainfall.Leptospirosis is characterized by hemorrhage, jaundice, myalgia, renal impairment, and aseptic meningitis [1,2].Pulmonary diffuse hemorrhage (PDH), a serious clinical type of leptospirosis, results in death in a quarter of affected patients [3].The genus Leptospira consists of a diverse group of pathogenic and saprophytic spirochetes which, based on the antigenic structure, are classified into different serovars [1].A universal feature of pathogenic leptospires is their ability to parasitize the proximal renal tubules of a wide variety of wild and domestic animals.Infection with hostadapted leptospiral serovars can result in lifelong renal carriage and urinary shedding.In humans, exposure to infected host animals or contaminated water or soil results in this potentially lethal disease [4].Leptospirosis eradication is difficult because there is an abundance of animal reservoirs, both wild and domestic, of Leptospira spp.and the long-term survival of the bacteria in the environment.Avoiding contact with animals chronically infected with Leptospira spp.(reservoirs) or their environments, such as soil and water contaminated with animal urine or carcasses, is the most effective means of disease prevention.However, the measure is difficult to practice, especially in the countries where agriculture is the foremost activity and environmental sanitation is compromised [5].Protective immunity elicited by leptospiral lipopolysaccharide is generally serovarspecific [2].The current available whole-cell vaccines cannot provide cross-protection against infection with more than 250 different Leptospira serovars known to exist and may lead to incomplete, short-term immunity and serious side effects.Despite vaccination, the disease still exists in some parts of the country [6][7][8].The design and construction of an efficient recombinant vaccine for leptospirosis control is very important [9,10].
Characterization of leptospiral outer membrane proteins (OMPs) has emerged as an important approach [11].
Outer membrane proteins of pathogenic bacteria species are very stable and are the foundation of communication between bacteria and the host.Therefore, many studies about the characteristics of membrane protein components have been performed.
OmpL1 is a surface genus-specific antigen of pathogenic Leptospirs that is not observed in the nonpathogenic species of Leptospira.This porin protein is significantly expressed during infection, demonstrating that it is an immunogenic antigen.OmpL1 can be a suitable candidate to be used in the preparation of a recombinant leptospirosis vaccine and in comprehensive serological diagnostic tests [12].
However, the diversity of ompL1 gene sequences from different pathogenic Leptospira spp.and the distribution of the ompL1 gene in vaccinal and clinical isolates have not been characterized.
In this study, for the first time, we sequenced and analyzed ompL1 genes cloned from standard pathogenic strains of leptospires prevalent in Iran.

Database
The ompL1 sequence of pathogenic Leptospira serovars were obtained from GenBank at the National Centre for Biotechnology Information (NCBI) website.Homology searches with the ompL1 sequences of different pathogenic Leptospira species were accomplished using the BLAST program against the GeneBank/NCBI nuclear acid sequence database.

Bacterial strains and media
The Microbiology Department of Razi Vaccine and Serum Research Institute in Karaj, Iran provided three standard strains belonging to three serogroups of pathogenic L. interrogans and one saprophytic strain of L. biflexa (Table 1).

Isolation of leptospiral genomic DNA
The culture was centrifuged at 17,000 x g at 4°C for 20 minutes.Genomic DNA was extracted from whole cells of samples grown to log phase growth using the phenol-chloroform extraction method [15].The preparation was added with a lysis buffer containing 10% SDS, EDTA (50mM), NaCl (100mM), Tris (100mM), 3 μL of 20 μg/mL proteinase-K, 5 μL of 20 mg/ml RNase A and incubated at 37°C for one hour.An equal volume of phenol-chloroform-isoamyl was added to the mixture.The preparation was centrifuged at 12,000 x g at 25°C for 5 minutes.The top phase was transferred to a new tube and isopropanol was added to precipitate the DNA.The DNA pellet was collected after centrifugation, washed with 70% ethanol, and airdried.The dried DNA was dissolved in TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA).The density and purity of the extracted DNAs were detected by UV spectrophotometery and analyzed by 1% agarose gel electrophoresis, ethidium bromide staining.

Primer design and ompL1 amplification by polymerase chain reaction
The oligonucleotide primers for amplification of the ompL1 gene by polymerase chain reaction (PCR) were designed from the DNA sequences encoding OmpL1 of L. interrogans strains of the GenBank database.Primer syntheses were performed at (SIGMA ALDRICH, Taufkirchen, Munich, Germany).
A gradient PCR was performed for determining the optimum primer annealing temperature.The temperature gradients were 47-58°C.
In order to provide appropriate amplicons for the TAclone method, a 50 μL PCR amplification reaction

Cloning of ompL1 amplicon into vectors and selection of E. coli transformants
To obtain more accurate sequence data, the ompL1 amplicons were purified using the GeneJET PCR Purification Kit (Thermo Scientific-Fermentas, Waltham, USA).The purified DNA was ligated into pTZ57R/T using the InsTAclone PCR Cloning Kit (Thermo Scientific-Fermentas, Waltham, USA) via the overhang T of the plasmid and A of the DNA.The recombinant vector was cloned into Top10 E. coli competent cells using heat shock protocol [15,16].
Selected transformed E. coli colonies (examined by Colony PCR using ompL1 specific primers) were individually inoculated into LB-ampicillin (50 μg/mL) broth.The cultures were incubated in a shaking incubator at 37°C for 16 hours.Bacterial cells were collected from each culture by centrifugation at 5,000 x g at 25°C for 5 minutes.Plasmids were extracted from the cell preparations using a Plasmid Isolation Kit (CinnaGen, Tehran, Iran).The purified plasmid preparation was analyzed by 1% agarose gel electrophoresis, ethidium bromide staining, and visualization under a UV transilluminator.Furthermore, PCR using ompL1-specific primers was performed to confirm the accuracy of recombinant purified plasmids.

Nucleotide sequencing and homological analysis
The ompL1 genes from all three pathogenic standard serovars were sequenced by SeqLab Co. (Germany).Homological analysis was performed by BLAST against the nucleotide sequence database on the GenBank/NCBI website (http://www.ncbi.nlm.nih.gov/).Sequence alignment was performed with the Clustal W method [17] using MEGA5 software [18].
The amino acid sequences of proteins were deduced from the nucleotide sequences using the EditSeq program of DNAStar software.
Amino acid sequence alignments were carried out using the Clustal W method of MegAlign program of DNAStar software package (DNASTAR, Madison, USA).
Phylogenetic analysis was done with neighborjoining (NJ) optimality criteria of MEGA5 software and MegAlign program.

Isolation of leptospiral genomic DNA and ompL1 amplification by PCR
Genomic DNA encoding OmpL1 was successfully extracted from pathogenic and saprophitic serovars, as the ompL1 could be amplified from the genomic DNA preparation.The DNA was used as a template for amplification of ompL1 by gradient PCR and the annealing temperature at 52°C was chosen.The ompL1 amplicon at 963 bp is shown in Figure 1.
All the three standard strains of pathogenic L. interrogans carried the ompL1 gene, since amplicons with the expected size were produced by PCR from these strains.However, saprophitic L. biflexa strains could not be amplified by PCR [11] (Figure 1).

Cloning, sequencing and homological analysis of the ompL1 gene
The ompL1 gene was successfully subcloned into pTZ57R/T vector and the recombinant vector, ompL1-pTZ57R/T, was introduced into competent Top10 E. coli.These colonies were then separately grown in LBampicillin broth and the plasmids were extracted and purified.The purified plasmid preparation was analyzed by 1% agarose gel electrophoresis and ethidium bromide staining.Furthermore, PCR using ompL1-specific primers was performed to confirm the accuracy of recombinant purified plasmids (data not shown).
The ompL1 genes of the three pathogenic Leptospira serovars were all 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 strain Lai [19], L. interrogans serovar Copenhageni strain Fiocruz L1-130 [20,5], the corresponding sequence data of Leptospira kirschneri serovar Grippotyphosa strain RM52 [21] and ompL1 sequences belonging to other pathogenic Leptospira strains. of the ompL1 gene sequence of three standard serovars was also performed.The results showed that sequences of the ompL1 gene of L. Canicola (RTCC2805) and L. Sejroe hardjo (RTCC2821) (Table 1) were exactly the same (100% identity), while L. Grippotyphosa (RTCC2808) was less similar (88.5%) to the other two serovars (Figures 2 and 4A).

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 [25,26].
Surface is a key characteristic for an effective antigen.OmpL1 is an immunogenic transmembrane porin protein extensively expressed in pathogenic leptospires [27,22].
According to earlier reports, a strong interaction between the recombinant OmpL1 protein and leptospirosis patients' sera was observed by enzymelinked immunosorbent assay.It has also been demonstrated that OmpL1 and LipL41 together could provide significant protection against homologous challenge in the hamster model of leptospirosis [26].
Moreover, a previously designed OmpL1 DNA vaccine was found to be well tolerated by the immunized animals and conferred immunity that protected some immunized hamsters against the heterologous lethal challenge.The vaccine was shown to confer the delay in the death time and reduced morbidity in the vaccinated animals [5].
OmpL1 should therefore be viewed as a potential candidate of genus-specific antigen for the development of new universal vaccines and serodiagnostic methods for leptospirosis in Iran.
We evaluated probable differences of the ompL1 gene and its protein product between various local pathogenic serovars.We sequenced and analyzed ompL1 genes cloned from standard pathogenic strains of leptospires prevalent in Iran and then compared the OmpL1 amino acid sequences.
According to our alignment and phylogenetic analysis from the three standard strains of pathogenic L. interrogans, although the ompL1 gene nucleotide sequence was slightly different within some of the strains, amino acid alignment (Figures 3 and 4B) of the OmpL1 proteins revealed that there was little difference among them.

Conclusion
The differences in nucleotide sequences in the ompL1 gene types may not affect the immunogenicity of OmpL1 proteins.The cloned gene, therefore, could be further used for expression; recombinant OmpL1 as an efficient and conserved antigen may be a useful vaccine candidate against leptospirosis in Iran.

Figure 1 .
Figure 1.PCR amplification of Leptospira species based on ompL1 gene

Figure 2 .
Figure 2. Nucleotide alignment of three pathogenic serovars performed with Clustal W method using MEGA5 software.

Figure 3 .
Figure 3. Amino acid alignment of three pathogenic serovars performed with Clustal W method using MegAlign program of DNAStar software package

Figure 4 .
Figure 4. Phylogenetic trees of three standard strains using the NJ method

Table 1 .
Leptospira strains included in this study