Mapping the antigenicity of the parasites in Leishmania donovani infection by proteome serology

Abstract

Background: Leishmaniasis defines a cluster of protozoal diseases with diverse clinical manifestations. The visceral form caused by Leishmania donovani is the most severe. So far, no vaccines exist for visceral leishmaniasis despite indications of naturally developing immunity, and sensitive immunodiagnostics are still at early stages of development.

Methodology/principle findings: Establishing a proteome-serological methodology, we mapped the antigenicity of the parasites and the specificities of the immune responses in human leishmaniasis. Using 2-dimensional Western blot analyses with sera and parasites isolated from patients in India, we detected immune responses with widely divergent specificities for up to 330 different leishmanial antigens. 68 antigens were assigned to proteins in silver- and fluorochrome-stained gels. The antigenicity of these proteins did not correlate with the expression levels of the proteins. Although some antigens are shared among different parasite isolates, there are extensive differences and no immunodominant antigens, but indications of antigenic drift in the parasites. Six antigens were identified by mass spectrometry.

Conclusions/significance: Proteomics-based dissection of the serospecificities of leishmaniasis patients provides a comprehensive inventory of the complexity and interindividual heterogeneity of the host-responses to and variations in the antigenicity of the Leishmania parasites. This information can be instrumental in the development of vaccines and new immune monitoring and diagnostic devices.

Figure 1. Crude extract of Leishmania donovani AG83 cells was separated in a 12% SDS-PAGE.

After transfer of the protein onto nitro cellulose each lane was incubated separately with serum of 15 individuals with symptoms of VL (lanes 1–15) and 4 healthy donor controls (lanes 16–19).

Figure 2. 2D-Western blot analysis of the serospecificities of different VL patients and of the antigenicities of L. donovani isolates from different endemic districts in Bihar, India.

Whole protein extracts of L. donovani BHU2 (MHOM/IN/02/BHU2) (Panels A–D) isolated from a patient from Motihari or BHU17 (MHOM/IN/02/BHU17) (Panels E–F) isolated from a patient from Muzaffarpur district were separated by 2-dimensional electrophoresis (pH-range of first dimension: 3–10 on 18 cm IPG-strips; second dimension: 12.5% SDS-PAGE). The separated proteins were blotted onto nitrocellulose membrane and probed with sera of different patients: A: patient No. 17; B: patient No. 2; C: patient No. 3; D: patient No. 14; E: patient No. 17; F: patient No. 3. Patients 2 and 3 were from Motihari, patients 14 and 17 from Muzaffarpur. For comparison of the serospecificities shown with Panels A–D, BHU2 proteins were probed with 4 different sera, 2 from Motihari, 2 from Muzaffarpur district. Dotted circles indicate antigens recognized by all 4 sera. Private specificities are not marked. For comparison of the antigenicities of different LD isolates shown with Panels C–F, proteins of BHU2 (Panels C and D) and BHU17 (Panels E and F) were probed with a serum from the same district and a serum from the respective other district. Circles with full lines in panels C–F indicate antigens that are detected only in one of the two LD isolates but not in the other.

Figure 3. Mapping of the serospecificities of VL patients against L. donovani antigens.

The proteins of total lysates of L. donovani isolate BHU2 (MHOM/IN/02/BHU2) promastigotes were separated by 2-dimensional gel electrophoresis with a pH gradient of 4.5–7 in the first dimension and a 12.5% SDS-PAGE in the second. A: Western blot developed with autologous serum. 330 Leishmania antigens could be counted. B: The corresponding silver-stained gel displaying 1067 protein spots. 68 of 330 antigens could be assigned to protein spots in the silver-stained gel. Six of the protein spots, indicated by arrows and numbered 1–6, were processed for protein identification by mass spectrometry. C: Inner section of a SyPro Ruby-stained gel with the same protein material. The proteins that match antigens detected by the Western blot analysis shown in Panel A are circled. 54 antigenic proteins are thus marked, 14 antigenic proteins are outside the sections shown. The arrows again indicate the six identified proteins.

Figure 4. Identification of L. donovani antigens by mass spectrometry.

The protein spots in the silver-stained gel that had been assigned to antigen spots in the corresponding Western blot were excised, destained and incubated with trypsin. The resulting fragments were extracted from the gel pieces and analyzed by MALDI-TOF-MS. Panels A–F show the peptide mass fingerprints (PMF) of the proteins in spots 1–6, respectively. Upon processing via MASCOT, the antigens were identified as HSP70 (spots 1, panel A and spot 2, panel B), gp63 (spot 3, panel A), EIF-4a (spot 4, panel C), Ef2 (spot 5, panel D) and grp78 (spot 6, panel E). The fragment masses that could be matched to theoretical trypsin digests of the identified proteins are indicated by asterisks. Open circles indicate the autolytic fragments of trypsin that were used for internal calibration.

Figure 5. Confirmation of the protein identifications for spots 4 and 5 by PSD mass spectrometry for peptide fragmentation fingerprint (PFF) analyses.

Panel A shows the PSD spectrum of a 1447 Da fragment of the protein in spot 4 and panel B that of a 1757 Da fragment from antigen spot 5. The former was identified as the tryptic fragment HNLIQGLVLSPTR from EIF-4a corresponding to the L. major sequence of this protein and the latter as the tryptic fragment AYLPVAESFGFTADLR of EF2 also of L. major. In both cases, the protein identification by PFM analyses were, thus, confirmed by PFF analyses and homology to the sequences of the proteins in L. major.