Epitope mapping of LipL32 protein of pathogenic leptospiras

LOTTERSBERGER, JAVIER; VANASCO, NORMA BIBIANA; VIARENGO, GASTÓN; GUERRERO, SERGIO ADRIÁN; FRANK, RONALD; TONARELLI, GEORGINA

Quito, Ecuador

Congreso; 5th Meeting of the International Leptospirosis Society (ILS); 2007

International Leptospirosis Society (ILS)

LipL32 is one of the most antigenic outer membrane proteins from pathogenic leptospiras. It has been reported the usefulness of this lipoprotein in the serodiagnosis of leptospirosis. In order to identify LipL32 minimum regions for antibody recognition, an epitope mapping was carried out by means of a library of overlapping peptide fragments prepared by simultaneous and parallel solid phase peptide synthesis on derivatized cellulose membranes (SPOT synthesis). Eighty seven overlapping decapentapeptides corresponding to the complete sequence of Leptospira interrogans serovar Copenhageni LipL32 (http://aeg.lbi.ic.unicamp.br/world/lic/) were synthesized. Each peptide sequence was offset by three amino acids from the previous peptide. Bound antibodies were detected with alkaline phosphatase (AP)-conjugated secondary antibody followed by a colour reaction with BCIP and MTT. In addition LipL32 was expressed heterologously in Escherichia coli and purified to homogeneity by chromatography. The recombinant protein, expressed as a fusion to the maltose binding protein (MBP) was evaluated by dot blot and western blot assays. All immunoassays were performed using human and rabbit positive and negative sera. Non specific MBP reaction was discarded. According to the spots-image intensities the most reactive regions were localized by spots 51 to 55 (region 151-177, AAKAKPVQKLDDDDDGDDTYKEERHNK) and spots 61 to 64, (region 181-204, LTRIKIPNPPKSFDDLKNIDTKKL). These results strongly suggest the existence of two antigenic regions within LipL32, which can be recognized by antileptospiral antibodies. These LipL32 reactive regions are highly conserved among antigenically variants of Leptospira spp. isolates and do not show homology with sequences from other microorganisms. MBP-LipL32 evidenced high reactivity in both immunoassays and high affinity for specific antibodies in dot blot assays. The recombinant protein showed higher reactivity than the peptides in the immunoassays, and may indicate the existence of discontinuous epitopes in LipL32 that might be relevant for diagnosis. Thus, we have identified two peptide sequences from Leptospira interrogans LipL32 which were recognized by specific antibodies. This finding has potential relevance not only for serodiagnosis but also for a start point in the characterization of targets for vaccine design.