The Leishmania infantum PUF proteins are targets of the humoral response during visceral leishmaniasis

Abstract

Background: RNA-binding proteins of the PUF family share a conserved domain consisting of tandemly repeated 36-40 amino acid motifs (typically eight) known as Puf repeats. Proteins containing tandem repeats are often dominant targets of humoral responses during infectious diseases. Thus, we considered of interest to analyze whether Leishmania PUF proteins result antigenic during visceral leishmaniasis (VL).

Findings: Here, employing whole-genome databases, we report the composition, and structural features, of the PUF family in Leishmania infantum. Additionally, the 10 genes of the L. infantum PUF family were cloned and used to express the Leishmania PUFs in bacteria as recombinant proteins. Finally, the antigenicity of these PUF proteins was evaluated by determining levels of specific antibodies in sera from experimentally infected hamsters. The Leishmania PUFs were all recognized by the sera, even though with different degree of reactivity and/or frequency of recognition. The reactivity of hamster sera against recombinant LiPUF1 and LiPUF2 was particularly prominent, and these proteins were subsequently assayed against sera from human patients. High antibody responses against rLiPUF1 and rLiPUF2 were found in sera from VL patients, but these proteins resulted also recognized by sera from Chagas' disease patients.

Conclusion: Our results suggest that Leishmania PUFs are targets of the humoral response during L. infantum infection and may represent candidates for serodiagnosis and/or vaccine reagents; however, it should be kept in mind the cross-reactivity of LiPUFs with antibodies induced against other trypanosomatids such as Trypanosoma cruzi.

Figure 1

Diagrammatic representation of structural motifs found in the L. infantum PUF proteins. The positions of the Puf repeats and the PUM-HD domain are indicated.

Figure 2

Recognition of the L. infantum PUF proteins by sera from experimentally infected hamsters. Sera (n = 10) were individually tested for reactivity to rLiPUF1-10, rHSP70 and SLA. C-labeled lines represent the cut-off value, calculated as the mean plus 3 SD of the values for pre-infection sera. Relative absorbance = absorbance value of test serum ÷ mean of the absorbance of sera from animals before infection (pre-inmune sera).

Figure 3

Analysis of IgG reactivity against rLiPUF1 and rLiPUF2. (A) The reactivity of sera from the experimentally infected hamsters was determined by ELISA against rLiPUF1, rLiPUF2 and a mixture of both. (B) ELISA reactivity of sera from patients with VL (n = 10) or Chagas' disease (Cha, n = 5) and healthy controls against rLiPUF1 and rLiPUF2. Cut-off value = 0.49.