Identification of Proteins in Promastigote and Amastigote-like Leishmania Using an Immunoproteomic Approach

Abstract

BackgroundThe present study aims to identify antigens in protein extracts of promastigote and amastigote-like Leishmania (Leishmania) chagasi syn. L. (L.) infantum recognized by antibodies present in the sera of dogs with asymptomatic and symptomatic visceral leishmaniasis (VL).Methodology/Principal FindingsProteins recognized by sera samples were separated by two-dimensional electrophoresis (2DE) and identified by mass spectrometry. A total of 550 spots were observed in the 2DE gels, and approximately 104 proteins were identified. Several stage-specific proteins could be identified by either or both classes of sera, including, as expected, previously known proteins identified as diagnosis, virulence factors, drug targets, or vaccine candidates. Three, seven, and five hypothetical proteins could be identified in promastigote antigenic extracts; while two, eleven, and three hypothetical proteins could be identified in amastigote-like antigenic extracts by asymptomatic and symptomatic sera, as well as a combination of both, respectively.Conclusions/SignificanceThe present study represents a significant contribution not only in identifying stage-specific L. infantum molecules, but also in revealing the expression of a large number of hypothetical proteins. Moreover, when combined, the identified proteins constitute a significant source of information for the improvement of diagnostic tools and/or vaccine development to VL.

Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

“…After database searches were performed in this study, eight antigens were identified as Leishmania-specific proteins. Since they were recognized by sera from L. infantum-infected dogs in a previous immunoproteomics study (29), their primary sequences were mapped to obtain B-cell-specific epitopes, which were synthesized and subsequently evaluated as new and more-refined antigens for a more sensitive and specific serodiagnosis of L. infantum infections in dogs.…”

“…In an attempt to identify more-refined antigens for the serodiagnosis of CVL, 26 hypothetical proteins from L. infantum, which were identified previously by means of an immunoproteomic approach (29), were evaluated in this study. Of the hypothetical leishmanial proteins investigated, eight were identified as Leishmania-specific proteins.…”

Recent updates and perspectives on approaches for the development of vaccines against visceral leishmaniasis

“…Immuno-proteomic approaches have been developed to identify new Leishmania proteins with distinct biological functions, such as new diagnostic markers, vaccine candidates, and/or potential drug targets (80) (81) (82) . The use of antileishmanial antibodies obtained from infected mammalian hosts contributed to the refinement of these analyses, by assisting in the identifi cation of antigens recognized by the immune system during active disease (82) (83) .…”

“…The use of antileishmanial antibodies obtained from infected mammalian hosts contributed to the refinement of these analyses, by assisting in the identifi cation of antigens recognized by the immune system during active disease (82) (83) . Immuno-proteomic approaches usually involve protein preparation and separation by bidimensional electrophoresis, followed by immunoblotting experiments, and subsequent identifi cation of protein spots by mass spectrometry (Figure 1).…”

“…The authors speculated about the use of these candidates as vaccines in future assays, owing to the existence of putative-T cell motifs in the antigens. In another immuno-proteomic approach, developed using protein extracts from the stationary promastigote and amastigote-like stages of L. infantum, several specific promastigote ( Table 2) and amastigote ( Table 3) hypothetical proteins were identifi ed in serum samples from dogs with asymptomatic and/or symptomatic VL (82) .…”