Prolyl Oligopeptidase From Leishmania infantum: Biochemical Characterization and Involvement in Macrophage Infection

Abstract

Leishmania infantum is a flagellated protozoan and one of the main causative agents of visceral leishmaniasis. This disease usually affects the human reticuloendothelial system, can cause death and available therapies may lead to serious side effects. Since it is a neglected tropical disease, the incentives for the development of new drugs are insufficient. It is important to know Leishmania virulence factors that contribute most to the disease in order to develop drugs. In the present work, we have produced L. infantum prolyl oligopeptidase (rPOPLi) in Escherichia coli, and investigated its biochemical properties as well as the effect of POP inhibitors on its enzymatic activity and on the inhibition of the macrophage infection by L. infantum. The optimal activity occurred at pH 7.5 and 37°C in the presence of DTT, the latter increased rPOPLi catalytic efficiency 5-fold on the substrate N-Suc-Gly-Pro-Leu-Gly-Pro-AMC. The enzyme was inhibited by TPCK, TLCK and by two POP specific inhibitors, Z-Pro-prolinal (ZPP, IC₅₀ 4.2 nM) and S17092 (IC₅₀ 3.5 nM). Besides being a cytoplasmic enzyme, POPLi is also found in punctuate structures within the parasite cytoplasm or associated with the parasite plasma membrane in amastigotes and promastigotes, respectively. Interestingly, S17092 and ZPP prevented parasite invasion in murine macrophages, supporting the involvement of POPLi in the invasive process of L. infantum. These data suggest POPLi as a virulence factor that offers potential as a target for designing new antileishmanial drugs.

Keywords: POPLi; drug target; leishmaniasis; protease; virulence factor.

FIGURE 1

SDS-PAGE and western blot of purified rPOPLi. Analysis of recombinant POPLi expressed in E. coli. (A) SDS-PAGE of fractions from flow-through, wash and elution steps (12 μL/lane). The gel was subjected to Coomassie Blue staining. Ext, total extract; FT, flow through; W1-W3, washes; Elu, elution fraction. (B) Western blot of purified rPOPLi (30 ng) and total protein extracted from 5 × 10⁶ L. infantum promastigotes, obtained after freezing and thawing, were performed with the anti-rPOPLi serum (1:200). POPLi predicted molecular weight is ∼78 kDa, and rPOPLi is ∼80 kDa.

FIGURE 2

Effects of different substrates, pH values, temperatures, additives and anti-POPLi antibodies on rPOPLi activity. (A) Substrates (20 μM final concentration) were tested in 25 mM HEPES pH 7.5 containing 150 mM NaCl and 5 mM DTT. Substrates: P (Pro-AMC), N-IA (N-Ile-Ala-AMC), Suc-GP (Suc-Gly-Pro-AMC), AAFA (Ala-Ala-Phe-AMC), RR (Arg-Arg-AMC), GP (Gly-Pro-AMC) and Suc-GPLGP (N-Suc-Gly-Pro-Leu-Gly-Pro-AMC). (B) Effect of temperature on rPOPLi activity. Activities were determined in pH 7.5 with incubation temperatures ranging from 28 to 45°C. (C) Stability of rPOPLi at different pH values. Activities were determined in AGMT buffer adjusted to different pHs. (D) Effect buffer (HEPES 25 mM or Tris 50 mM), salt (0, 150, or 300 mM NaCl) and reducing agent DTT (0, 5, or 10 mM) on rPOPLi activity. (E) Substrate concentration on rPOPLi activity and determination of enzyme kinetic parameters in the presence or not of DTT. The assays were performed in triplicate. *p < 0.05 and ****p < 0.0001.

FIGURE 3

Leishmania infantum viability was not affected by POPLi inhibition. (A) POPLi inhibition by specific POP inhibitors ZPP and S17092. (B) POPLi inhibition by anti-POPLi serum. (C) Effect of POP inhibitors on L. infantum viability. Promastigotes (4 × 10⁶ cells/mL) were incubated for 48 h in the presence of 12.5 to 200 μM ZPP or S17092, or 0.1% DMSO (control). Viability was determined using Resazurin. Error bars represent standard deviations of triplicate samples. (D) ZPP and S17092 can penetrate L. infantum cell membrane. Promastigotes were incubated in the presence of either ZPP or S17092 for 1 h. Parasites were washed with PBS and lysed by freezing/thawing. Protein total extract was tested against N-Suc-Gly-Pro-Leu-Gly-Pro-AMC. (E) Enzymatic activity of other proteases is not affected by ZPP or S17092. Promastigote cell lysate was tested against Gly-Pro-AMC (dipeptidyl peptidases), Z-Gly-Gly-Arg-AMC (oligopeptidase B) and Suc-Leu-Leu-Val-Tyr-AMC (cathepsin B). **p < 0.01 and ***p < 0.001.

FIGURE 4

POPLi localization and enzymatic activity in parasite extract fractions. (A) 1 × 10⁷ L. infantum promastigotes were lysed by freeze and thaw cycles to produce a soluble cytoplasmic fraction (CTPL) and an insoluble fraction, which was solubilized with Triton X-100 to produce an insoluble cytoskeleton fraction (CSKL) and a soluble membrane bound protein fraction (MEMB). Native POPLi expression in all subcellular fractions was analyzed by Western blotting using anti-rPOPLi serum followed by ECL detection. rPOPLi (80 ng) was loaded for reference. As sample loading control, the same membrane was probed with the anti-alpha tubulin TAT1 monoclonal antibody IgG2b (1:2000). (B) Enzymatic activity against Suc-Gly-Pro-Leu-Gly-Pro-AMC was measured in total fraction after BugBuster lysis (Total) and in the CTPL, CSKL and MEMB fractions obtained after freezing and thawing lysis. *p < 0.05, Student’s t-test.

FIGURE 5

Cellular distribution of POPLi in the developmental forms of Leishmania infantum. Axenic amastigotes and promastigotes were fixed in 1% formaldehyde/PBS and stained with POPLi antiserum followed by incubation with Alexa Fluor 488-conjugated anti-mouse IgG (green). Parasite DNA was stained with DAPI (red). Overlay of fluorescence images.

FIGURE 6

POP specific inhibitors reduce macrophage infection by Leishmania infantum. Murine macrophages were infected with L. infantum promastigotes (MOI = 10:1) after parasite incubation with POP inhibitors (A) Z-Pro-Prolinal (ZPP) and (B) S17092. (C) Murine macrophages were pre-incubated 1 h with POP inhibitors before infection. (D) Infection assay in the presence of rPOPLi (boiled or not boiled). (E) Infection assay after 30 min treatment of promastigotes with anti-rPOPLi serum. (F) Infection assay in the presence of POP immune complexes: rPOPLi (10 μg) and anti-POPLi antiserum pre-incubation for 30 min to form immune complexes that were added along with the parasites in the infection assay. None: neither promastigotes nor macrophages were submitted to any pre-treatment. Assays were performed in triplicate. Infection was evaluated by percentage of infected cells compared to the control. *p < 0.05 and ****p < 0.0001.