Cloning, characterization, and serodiagnostic evaluation of Leishmania infantum tandem repeat proteins

Abstract

Visceral leishmaniasis (VL) is a form of leishmaniasis, which is caused by infection with the protozoan parasite Leishmania, and is often fatal unless it is treated. Rapid and accurate diagnosis of VL is important for effective treatment. Here we report the cloning of previously undescribed tandem repeat (TR) proteins of Leishmania infantum and an evaluation of VL patient antibody responses to the corresponding proteins. By screening an L. infantum expression library with sera from human VL patients or infected hamsters, we identified 43 genes encoding B-cell antigens. Surprisingly, 19 of the 43 genes (44%) were TR proteins, and that percentage was significantly higher than that for genes picked randomly from the database. We then expressed the TR regions of LinJ16.1750, LinJ22.1590, and LinJ33.2870 and the entire LinJ28.2310 protein. These recombinant proteins were all recognized by Sudanese VL patient sera in an enzyme-linked immunosorbent assay. Recombinant LinJ16.1750 (rLinJ16.1750) showed the best performance among these antigens in terms of both sensitivity and specificity. Serological evaluation revealed that 97% (34 of 35) of Sudanese VL patients had significantly elevated antibody levels to rLinJ16.1750. Furthermore, when eight of the patient sera which had low reactivities to rK39 were tested with the novel recombinant antigens, some of the sera showed stronger antibody responses to these antigens than to rK39. Our results suggest that TR regions from the novel L. infantum proteins identified in this study are immunodominant B-cell epitopes and may represent good candidates for serodiagnosis of VL.

FIG. 1.

PCR analysis of TR genes. PCRs were performed with primer sets specific for the TR regions of LinJ16.1750, LinJ22.1590, and LinJ33.2870 and for a T. cruzi gene, using total DNAs of L. infantum (Li), L. donovani (Ld), L. major (Lm), L. amazonensis (La), and T. cruzi (Tc) as templates. Sizes are shown in base pairs.

FIG. 2.

Expression and purification of L. infantum recombinant proteins. The images show Coomassie blue-stained sodium dodecyl sulfate-4 to 20% polyacrylamide gradient gels containing uninduced E. coli lysates (lanes 1), induced lysates (lanes 2), and purified proteins (lanes 3). Sizes are shown in kDa. The apparent molecular masses of the proteins were comparable to their predicted molecular masses.

FIG. 3.

ELISA evaluation of patient seroreactivities to L. infantum recombinant proteins and LiSLA. Sera from patients with visceral leishmaniasis (VL, n = 10), cutaneous leishmaniasis (CL, n = 10), tuberculosis (TB, n = 10), and malaria (n = 6) and sera from healthy controls in the United States (n = 10) were used. The mean and standard error of the mean of the OD values for each group are shown.

FIG. 4.

Reactivities of human VL patient sera to TR proteins. (A) Sera from VL patients (n = 35) and healthy controls (n = 20) were tested for reactivity to rLinJ16.1750r2 or rK39 by ELISA. The mean for each group is shown as a solid line. Dotted lines represent the cutoff value, calculated as the mean plus 3 SD of the OD values for healthy controls. (B) Recognition of recombinant proteins by sera from eight VL patients which showed low reactivities to rK39 (ODs of <1.0 [circled in panel A]).