Characterization of Leishmania (L.) amazonensis oligopeptidase B and its role in macrophage infection

Abstract

Leishmania spp. are parasitic protozoa that cause leishmaniasis, a disease endemic in 98 countries. Leishmania promastigotes are transmitted by the vector and differentiate into amastigotes within phagocytic cells of the vertebrate host. To survive in multiple and hostile environments, the parasite has several virulence factors. Oligopeptidase B (OPB) is a serine peptidase present in prokaryotes, some eukaryotes and some higher plants. It has been considered a virulence factor in trypanosomatids, but only a few studies, performed with Old World species, analysed its role in Leishmania virulence or infectivity.L. (L.) amazonensis is an important agent of cutaneous leishmaniasis in Brazil. The L. (L.) amazonensis OPB encoding gene has been sequenced and analysed in silico but has never been expressed. In this work, we produced recombinant L. (L.) amazonensis OPB and showed that its pH preferences, K_m and inhibition patterns are similar to those reported for L. (L.) major and L. (L.) donovani OPBs. Since Leishmania is known to secrete OPB, we performed in vitro infection assays using the recombinant enzyme. Our results showed that active OPB increased in vitro infection by L. (L.) amazonensis when present before and throughout infection. Our findings suggest that OPB is relevant to L. (L.) amazonensis infection, and that potential drugs acting through OPB will probably be effective for Old and New World Leishmania species. OPB inhibitors may eventually be explored for leishmaniasis chemotherapy.

Keywords: Enzyme activity; inhibition; macrophage infection; oligopeptidase B.

Graphical abstract

Fig. 1.

Recombinant L. (L.) amazonensis OPB expression and purification. (A) 10% SDS-PAGE showing soluble (S.F.) and insoluble (I.F.) fractions of bacterial extracts after sonication. Protein marker: SeeBlue Plus2, Invitrogen. (B) 10% SDS-PAGE showing fractions (Fr.) 1–5 of the soluble fraction of the bacterial lysate eluted from the nickel column. Protein marker: SeeBlue Plus2, Invitrogen.

Fig. 2.

Characterization of the enzymatic activity of the recombinant OPB from L. (L.) amazonensis. (A) Activity in different pHs. The substrate Z-Arg-Arg-AMC was prepared in 0.2 M citrate-phosphate and 50 mm Tris-HCl buffers. Mean relative activities and standard deviations were calculated based on 3 enzyme assays. For more details see Materials and Methods. Negative relative activities were considered as 0. (B) Activity in the presence of commercial inhibitors and bivalent cations. Inhibitors were pre-incubated with the enzyme for 10 min and the activity was measured by hydrolysis of the substrate Z-Arg-Arg-AMC in a 50 mm Tris-HCl buffer, pH 8.0. Negative relative activities were considered as 0. Mean relative activities and standard deviations were calculated based on 3 enzyme assays. For more details see Materials and Methods. Statistical analysis for one-way ANOVA and Sidak's multiple comparison (*P < 0.05; **P < 0.005; ****P < 0.0001).

Fig. 3.

Western blot to assess the presence of OPB in L. (L.) amazonensis promastigotes from days 2, 4 and 6 and amastigotes, in parallel with recombinant (Lama OPB). 20 μg of each total protein extract and 0.5 μg of recombinant protein were probed with the primary anti-OPB serum at a dilution of 1:500 and secondary anti-mouse HRP at a dilution of 1:5000. Protein marker: SeeBlue Plus2, Invitrogen

Fig. 4.

Infection of peritoneal macrophages with L. (L.) amazonensis in the presence of recombinant OPB (100 ng mL⁻¹), OPB inhibited with Pefabloc^® (Sigma) (OPB + Pefa, 100 ng mL⁻¹) and denatured OPB (Denat. OPB, 100 ng mL⁻¹) throughout the experiment. (A) Percentage of infected macrophages. (B) Number of amastigotes per macrophage. Results represented as mean ± standard deviation of 3 technical replicates from a representative experiment of 3 with similar profiles. Statistical analysis by one-way ANOVA and Tukey's multiple comparisons (*P ⩽ 0.01; **P ⩽ 0.005; ***P ⩽ 0.0005).

Fig. 5.

Infection of MDMOs with L. (L.) amazonensis in the presence of recombinant OPB (100 and 200 ng mL⁻¹), OPB inhibited with Pefabloc^® (Sigma) (OPB + Pefa 100 and 200 ng mL⁻¹) and denatured OPB (Denat. OPB, 100 and 200 ng mL⁻¹) throughout the experiment. (A) Percentage of infected macrophages. (B) Number of amastigotes per macrophage. Results of a single experiment, represented as mean ± standard deviation of 3 technical replicates. Statistical analysis by one-way ANOVA and Tukey's multiple comparisons.