Lopinavir and Nelfinavir Induce the Accumulation of Crystalloid Lipid Inclusions within the Reservosomes of Trypanosoma cruzi and Inhibit Both Aspartyl-Type Peptidase and Cruzipain Activities Detected in These Crucial Organelles.

Leandro S Sangenito,Marta H Branquinha,André L S Santos,Miria G Pereira,Thais Souto-Padron

doi:10.3390/tropicalmed6030120

Abstract

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite’s lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan.

Highlights

Human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) have been the cornerstone of HIV-infected individuals’ treatment since its advent in the mid-1990s.In addition to impairing the replication and maturation of viral particles [1], the introduction of HIV-PIs to highly active antiretroviral therapy (HAART) had an impact on opportunistic coinfections [2,3,4,5]
We reported the side effects caused by treatment with the HIV-PIs lopinavir and nelfinavir on the storage of high amounts of lipids in the reservosomes of T. cruzi epimastigotes by transmission electron microscopy, as well as the inhibition of both aspartyl-type peptidase and cruzipain activities detected within these isolated key organelles
It is clear that treatment with HIV-PIs somehow affects the lipid metabolism of T. cruzi, as occurs in the treatment of HIV-positive patients [7,8]

Summary

Introduction

In addition to impairing the replication and maturation of viral particles [1], the introduction of HIV-PIs to highly active antiretroviral therapy (HAART) had an impact on opportunistic coinfections [2,3,4,5]. In this line, our research group studied the repositioning of HIV-PIs against Trypanosoma cruzi, where the most effective PIs, lopinavir and nelfinavir, had a huge effect on all life forms of the parasite (for a comprehensive reading, see the review [6]). Similar alterations were reported by our research group in Leishmania amazonensis promastigotes [9,10], reflecting a common metabolic alteration promoted by HIV-PIs in both lower (e.g., trypanosomatids) and higher eukaryotic organisms

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