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 . 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 [21–24]. 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 [16–18]. 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 immuno-electron 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 typically used . 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 , 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.