Cruzipain Activates Latent TGF-β from Host Cells during T. cruzi Invasion.

Patrícia Mello Ferrão,Wim Maurits Degrave,Luciana Ribeiro Garzoni,Sabine Bailly,Claudia Masini D'Avila-Levy,Tania Cremonini Araujo-Jorge,Jean Jacques Feige,Leila Mendonça-Lima,Ana Paula Lima,Mariana Caldas Waghabi,Antônio Da Silva Gonçalves

doi:10.1371/journal.pone.0124832

Abstract

Several studies indicate that the activity of cruzipain, the main lysosomal cysteine peptidase of Trypanosoma cruzi, contributes to parasite infectivity. In addition, the parasitic invasion process of mammalian host cells is described to be dependent on the activation of the host TGF-β signaling pathway by T. cruzi. Here, we tested the hypothesis that cruzipain could be an important activator of latent TGF-β and thereby trigger TGF-β-mediated events crucial for the development of Chagas disease. We found that live epimastigotes of T. cruzi, parasite lysates and purified cruzipain were able to activate latent TGF-β in vitro. This activation could be inhibited by the cysteine peptidase inhibitor Z-Phe-Ala-FMK. Moreover, transfected parasites overexpressing chagasin, a potent endogenous cruzipain inhibitor, prevented latent TGF-β activation. We also observed that T. cruzi invasion, as well as parasite intracellular growth, were inhibited by the administration of Z-Phe-Ala-FMK or anti-TGF-β neutralizing antibody to Vero cell cultures. We further demonstrated that addition of purified cruzipain enhanced the invasive activity of trypomastigotes and that this effect could be completely inhibited by addition of a neutralizing anti-TGF-β antibody. Taken together, these results demonstrate that the activities of cruzipain and TGF-β in the process of cell invasion are functionally linked. Our data suggest that cruzipain inhibition is an interesting chemotherapeutic approach for Chagas disease not only because of its trypanocidal activity, but also due to the inhibitory effect on TGF-β activation.

Highlights

The flagellate protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a pathology characterized by chronic inflammation associated with cardiomyopathy, which affects approximately 10 million people worldwide [1]
Since a previous study has demonstrated that both trypomastigote and amastigote forms of T. cruzi are able to activate latent TGF-β [3], we first verified whether epimastigotes could activate latent TGF-β
As cruzipain is one of the main peptidases expressed by T. cruzi epimastigotes, we tested if purified cruzipain could directly activate latent TGF-β in vitro

Summary

Introduction

The flagellate protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a pathology characterized by chronic inflammation associated with cardiomyopathy, which affects approximately 10 million people worldwide [1] Cytokines such as TGF-β participate in important processes during the parasite’s life cycle. Cruzipain plays vital roles during T. cruzi life cycle: it helps in the penetration of trypomastigotes into host cells [19, 20], is crucial for metacyclogenesis and intracellular development [21], participates in the development of host immune response triggered by the parasite [22] and is involved in the interaction with the insect host [23]. We tested the hypothesis that cruzipain might be an important activator of latent TGF-β and that this activation might result in a strong biological response related to the process of T. cruzi host cell invasion. Our data demonstrate that the ability of cruzipain to favor host cell invasion by T. cruzi is dependent upon TGF-βactivation

Results

Discussion

Experimental Procedures