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, IC 50 4.2 nM) and S17092 (IC 50 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.

Discovery of Leishmania Druggable Serine Proteases by Activity-Based Protein Profiling

“…Interestingly, when transcript levels of the two proteins were analyzed (Fiebig et al, 2015), the levels of the carboxypeptidase LmxM.18.0450 fell on transformation from promastigote to amastigote, whereas expression of POP LmxM.36.6750 increased considerably, suggesting that this is a mechanism by which the parasite adapts to the host-cell environment. Consistent with this, other studies on the Trypanosoma cruzi orthologue POP Tc80 and, more recently, the L. infantum homolog have proposed that this enzyme may be important for degrading the extracellular matrix and thus allowing host-cell invasion (Bastos et al, 2005;Motta et al, 2019;Lasse et al, 2020). These observations, combined with our results, imply that this is a common role for this enzyme across other Leishmania spp.…”

“…However, ZPP has no observable antiparasitic properties against both promastigote (EC 50 > 100 µM) and axenic amastigote (EC 50 > 100 µM), suggesting that either the prolyl oligopeptidase (LmxM.36.6750) is redundant, and its activity can be performed by other proteases, or it has a role in the interaction with the human host cell. Support for the last possibility is provided by the observation that infection of a macrophage can be inhibited by addition of ZPP to the media, but that ZPP had no detectable impact on the untreated macrophage (Lasse et al, 2020). Interestingly, when transcript levels of the two proteins were analyzed (Fiebig et al, 2015), the levels of the carboxypeptidase LmxM.18.0450 fell on transformation from promastigote to amastigote, whereas expression of POP LmxM.36.6750 increased considerably, suggesting that this is a mechanism by which the parasite adapts to the host-cell environment.…”

“…This revealed that, consistent with its known broad-spectrum of activity, chymostatin exhibited very high cytotoxicity with an EC 50 = 26 + 6 nM in promastigotes and an EC 50 = 22 + 2 nM in amastigotes, suggesting that TAMRA-FP only identifies a small subset of its protein targets. In contrast, ZPP was inactive up to 100 µM in both life stages, suggesting that the prolyl oligopeptidase is either redundant or is involved in infection and the interaction with the host immune response, without compromising parasite survival (Lasse et al, 2020). Finally, lactacystin had an activity of EC 50 = 23 ± 4 μM in promastigotes and EC 50 = 12 ± 2 μM in amastigotes, suggesting that TAMRA-FP identifies at least one of the molecular targets of this antibiotic.…”

Three types of Leishmania mexicana amastigotes: Proteome comparison by quantitative proteomic analysis

“…A higher CPB expression in lesion-derived amastigotes could be also related to larger megasome volumes in amastigotes from the infected animal ( Ueda-Nakamura et al., 2007 ). Prolyl oligopeptidase family protein is another potential virulence factor upregulated in lesion-derived amastigotes compared to axenic and macrophage-derived ones, it seems to be involved in macrophage invasion process as found in L. infantum ( Lasse et al., 2020 ).…”

Effects of a Serine Protease Inhibitor N-p-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on Leishmania amazonensis and Leishmania infantum

“…TPCK is an irreversible serine protease inhibitor belonging to the chymotrypsin-like class that alkylates the His residue in the active site of serine proteases and has been widely applied in several parasite trials [11,18] and tested against other pathologies, such as cancer [19]. Although chymotrypsin is not present in the Leishmania genus [20], TPCK can inhibit other serine peptidases, as it has already been shown that TPCK can inhibit prolyl oligopeptidase of L. infantum [21]. In another study in which serine proteases were purified from L. amazonensis, a 68 kDa fraction was found to be 100% inhibited by TPCK [22].…”