Revisiting cysteine protease function in Trypanosoma cruzi: implications for parasite egress and differentiation

Chagas disease is a major global health concern affecting millions of people worldwide, with limited therapeutic options in its chronic phase and no prophylactic vaccine. The causative agent, Trypanosoma cruzi, is a unicellular eukaryotic parasite whose life cycle alternates between insect vectors and a wide range of mammalian hosts. In mammalian cells, parasite proliferation depends on iterative cycles of host cell invasion, intracellular multiplication, differentiation, and host cell rupture, releasing hundreds of infective parasites. The mechanisms governing the critical transition from replicative amastigotes to infective trypomastigotes (trypomastigogenesis) and subsequent egress remain poorly understood, largely due to the lack of robust analytical tools. Here, we combined real-time cellular impedance monitoring, stage-specific fluorescent parasites, ultrastructural expansion microscopy, and automated high-content imaging to dissect the late steps of the lytic cycle. We provide quantitative evidence that trypomastigogenesis is temporally coordinated with egress, ensuring the release of fully mature, infective trypomastigotes. Furthermore, we re-evaluate the effect of the cysteine protease inhibitor Z-Phe-Ala fluoromethyl ketone (Z-FA-FMK) at late stages of infection. Our results quantitatively support previous observations that Z-FA-FMK impairs trypomastigogenesis, causing an accumulation of amastigotes and blocking progression to mature trypomastigotes. This arrest delays egress and leads to the release of immature forms, highlighting the essential role of cysteine proteases in parasite differentiation. Together, our work establishes a quantitative framework for dissecting the tightly regulated, multi-step process of lytic cycle termination in T. cruzi and offers a versatile platform for phenotypic screening and drug discovery.

Chagas disease, caused by Trypanosoma cruzi, affects millions worldwide and remains a major global health burden, causing chronic cardiac, digestive, and neurological complications. The disease, disproportionately impacting vulnerable populations, lacks effective treatments for the chronic phase of the disease or a vaccine for its prevention. Parasite replication and host cell exit are tightly linked, but the mechanisms driving the transition from intracellular replicative parasites to infective forms and their subsequent release upon host cell lysis are poorly understood. Using real-time monitoring, fluorescent parasites, and high-resolution imaging, we provide quantitative evidence that cysteine proteases are critical for parasite maturation and that their inhibition uncouples differentiation from egress, leading to the release of immature parasites that are generally considered less infective. These findings reveal fundamental principles of parasite biology, provide a platform for drug discovery, and highlight new avenues to target Chagas disease at a critical stage of infection.