Upregulated expression of B-cell antigen family tandem repeat proteins by Leishmania amastigotes

Abstract

Proteins with tandem repeat (TR) domains have been found in various protozoan parasites, and they are often targets of B-cell responses. Through systematic analyses of whole proteomes, we recently demonstrated that two trypanosomatid parasites, Leishmania infantum and Trypanosoma cruzi, are rich in antigenic proteins with large TR domains. However, the reason that these proteins are antigenic was unclear. Here, by performing molecular, immunological, and bioinformatic characterizations of Leishmania TR proteins, we found two possible factors affecting the antigenicity of these proteins; one factor is their fundamental composition as TR proteins, and the other is regulation of their expression by parasites. Enzyme-linked immunosorbent assays (ELISAs) using recombinant proteins revealed that the copy number of the repeat affects the affinity of binding between antigens and antibodies, as expected based on thermodynamic binding kinetics. Other than containing TR domains, the TR proteins do not share characteristics, such as sequence similarity or biased cellular location predicted by the presence of a signal sequence(s) and/or a transmembrane domain(s). However, the TR proteome contained a higher percentage of proteins upregulated in amastigotes than the whole proteome, and upregulated expression of a TR protein seemed to affect its antigenicity. These results indicate that Leishmania parasites actively utilize the TR protein family for parasitism in mammalian hosts.

FIG. 1.

Construction of proteins with different numbers of repeats. (A) Diagrams of two 73-aa repeat genes, LmjF16.1600 from L. major and LinJ16_V31760 from L. infantum. (B) Deduced amino acid sequence of the entire TR domain and flanking 6-aa sequences of LinJ16_V31760. The fragment shown was PCR amplified and cloned as Li73r8.7. (C) Schematic diagrams of Li73 recombinant proteins. Li73r8.7 was composed of the entire amino acid sequence shown in panel B flanked by the same 20-aa His tag present at the N terminus in other recombinant proteins.

FIG. 2.

Copy number of the TR affects the affinity between antigen and antibody. (A) Single-copy 73-aa repeat proteins with different sequences (Li73r1, Li73r1′, and Li73r1") were examined by ELISA using sera from human VL patients (left) and healthy controls (right). Symbols connected by a line indicate ELISA results obtained with an individual plasma sample. (B) Proteins with different copy numbers of the 73-aa repeat (Li73r1, Li73r2, and Li73r8.7) were examined by ELISA, as described above for panel A. (C) Comparison of ELISA reactivities for 200 ng/well of Li73r1 and 50 ng/well of Li73r8.7. (D) Proteins with different copy numbers of the 39-aa repeat (39r2, 39r4, and the original rK39 protein containing 6.5 copies) were examined by ELISA as described above for panel A. OD, optical density. *, P < 0.05 by t test; **, P < 0.01 by t test.

FIG. 3.

Antibody responses to TR antigens are distinct from the antibody responses to non-TR antigens. (A) Total IgG responses of L. major-infected BALB/c mice to a crude antigen (LmSLA) and defined antigens (TSA, LmSTI1, Li73r2, and TR2). (B) Comparison of total IgG responses to the antigens for BALB/c and nude mice, both infected with L. major. (C) IgG subclasses of L. major-infected BALB/c mice for the four defined antigens. (D) IgG1/IgG2a ratios for individual antigens (D). OD, optical density; Ab, antibody; NS, not significant. **, P < 0.01 by t test.

FIG. 4.

Upregulated expression of L. infantum TR proteins in amastigotes. (A) Mean values for fold changes in levels of expression of all 1,712 proteins (ALL) and 28 L. infantum TR proteins (TR), both detected by iTRAQ. (B) Western blot analysis of expression of K39, K26, A2, Li73, and KMP11 by both promastigotes and amastigotes. Ama and A, amastigotes; Pro and P, promastigotes. *, P < 0.05 by t test; **, P < 0.01 by t test.

FIG. 5.

Relationship between the expression pattern of a protein and its antigenicity. (A) TR proteins which were detected by iTRAQ were analyzed to determine their antigenicity by ELISA using human VL patient sera. The data for individual proteins were plotted (•) based on the log₂ difference in expression between promastigotes (zero time) and amastigotes (144 h) using the iTRAQ data (x axis) and the mean optical density (OD) determined by the ELISA (y axis). For some proteins (LinJ20_V3.1210, LinJ26_V3.2180, LinJ27_V3.0250, LinJ32_V3.2370, and LinJ34_V3.2360) there were no iTRAQ values at 144 h (▪); the values for the last available time point were used in these cases (see Table S2 in the supplemental material). Pearson r and P values from a correlation test are shown. (B) The correlation test was performed again after exclusion of two outliers indicated by arrows in panel A. OD, optical density.