Discussion

Cutaneous leishmaniasis, is a disease complex with high incidence in Colombia, caused mainly by parasites of the species Leishmania panamensis. To date, an efffective vaccine for leishmaniasis is not available and drug treatments have many complications. Therefore, it is necessary to make a rational search of vaccine candidates and new drugs, which requires information of the genome and transcriptome of this parasite. The purpose of this study was to compare the transcriptome between amastigotes and promastigotes from L. panamensis to identify stage-specific expressed genes and new vaccine candidates. We found 123 up-regulated and 127 down-regulated transcripts in amastigotes. Comparative analysis with genomes of other species and in silico analysis allowed us to select twenty conserved genes highly immunogenic. The biological characterization and cell localization of two of these proteins, LPAL13_270024700 and LPAL13_220016330, were evaluated in both stages. The first one, was a hypothetical protein with a BAR domain that is located in the promastigotes flagellum and in the flagellar pocket of amastigotes. This protein was overexpressed in promastigotes, and showed higher expression in highly virulent L. panamensis strains. In the other side, the protein LPAL13_220016330 presented a MAK16-L28e family domain and a nuclear localization signal (NLS), with a conserved zinc motif and phosphorilation target to CK2 kinase. The expression analysis showed that the amastigotes have higher expression by qRT-PCR and Western blot. Furthermore, the protective efficacy of both proteins in J774 cells and mice was evaluated using recombinant proteins with and without encapsulation in MPGE-PLA nanoparticles, and DNA vaccines as antigens. Interestingly, the cells J774 pre-treated with LPAL13_270024700 protein decreased significantlly the number of intracellular amastigotes. Moreover, a high protection was observed in L. panamensis infected BALB/C mice. Thus, the results indicate that hypothetical proteins are promising source of new candidates to develop vaccines against leishmaniasis. Further studies to evaluate the possible mechanism of protection of these genes should be performed.