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

Abstract

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.

Figure 1

Maximum parsimony trees for ompL1 nucleotide sequences (A) and its amino acid sequences (B) of 15 standard strains. This figure shows the upper quartile, for the full image please see Additional file 1. The numbers at each fork node indicate the bootstrapping values (shown only when > 50%). RefSeq accession number and abbreviation for the organism are shown at each relevant branch. The scale bar indicates the number of character substitutions.

Figure 2

Comparison of the predicted secondary structures and antigenic index of OmpL1 proteins. This figure shows the upper quartile, for the full image please see Additional file 2. The related strains of the same gene type just show the same predicted structure topology. Thus, only the results from three representative strains are showed here.

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.

Figure 4

Recognition of purified rOmpL1 proteins by sera from leptospirosis patients. Lane 1: protein markers (BioColor); lanes 2–13: the rOmpL1/1, rOmpL1/2 and rOmpL1/3 hybridizing with the antisera from leptospirosis patients; lane 13: normal human serum used as control.

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.

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).