RESUMO
Among the neglected tropical diseases, leishmaniasis is one of the most devastating, resulting in significant mortality and contributing to nearly 2 million disability-adjusted life years. Cutaneous leishmaniasis is a debilitating disorder caused by the kinetoplastid protozoan parasite Leishmania major, which results in disfiguration and scars. L. major genome was the first to be sequenced within the genus Leishmania. Use of proteomic data for annotating genomes is a complementary approach to conventional genome annotation approaches and is referred to as proteogenomics. We have used a proteogenomics-based approach to map the proteome of L. major and also annotate its genome. In this study, we searched L. major promastigote proteomic data against the annotated L. major protein database. Additionally, we searched the proteomic data against six-frame translated L. major genome. In all, we identified 3613 proteins in L. major promastigotes, which covered 43% of its proteome. We also identified 26 genome search-specific peptides, which led to the identification of three novel genes previously not identified in L. major. We also corrected the annotation of N-termini of 15 genes, which resulted in extension of their protein products. We have validated our proteogenomics findings by RT-PCR and sequencing. In addition, our study resulted in identification of 266 N-terminally acetylated peptides in L. major, one of the largest acetylated peptide datasets thus far in Leishmania. This dataset should be a valuable resource to researchers focusing on neglected tropical diseases.
Assuntos
Leishmania major/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Ontologia Genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Leishmania major/genética , Leishmaniose Cutânea/parasitologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Doenças Negligenciadas/parasitologia , Proteoma/química , Proteoma/genética , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em TandemRESUMO
Visceral leishmaniasis or kala azar is the most severe form of leishmaniasis and is caused by the protozoan parasite Leishmania donovani. There is no published report on L. donovani genome sequence available till date, although the genome sequences of three related Leishmania species are already available. Thus, we took a proteogenomic approach to identify proteins from two different life stages of L. donovani. From our analysis of the promastigote (insect) and amastigote (human) stages of L. donovani, we identified a total of 22,322 unique peptides from a homology-based search against proteins from three Leishmania species. These peptides were assigned to 3711 proteins in L. infantum, 3287 proteins in L. major, and 2433 proteins in L. braziliensis. Of the 3711 L. donovani proteins that were identified, the expression of 1387 proteins was detectable in both life stages of the parasite, while 901 and 1423 proteins were identified only in promastigotes and amastigotes life stages, respectively. In addition, we also identified 13 N-terminally and one C-terminally extended proteins based on the proteomic data search against the six-frame translated genome of the three related Leishmania species. Here, we report results from proteomic profiling of L. donovani, an organism with an unsequenced genome.