ABSTRACT

Cruzipain, the major cysteine protease of the pathogenic protozoa Trypanosoma cruzi, is an important virulence factor that plays a key role in the parasite nutrition, differentiation and host cell infection. Cruzipain is synthesized as a zymogen, matured, and delivered to reservosomes. These organelles that store proteins and lipids ingested by endocytosis undergo a dramatic decrease in number during the metacyclogenesis of T. cruzi. Autophagy is a process that digests the own cell components to supply energy under starvation or different stress situations. This pathway is important during cell growth, differentiation and death. Previously, we showed that the autophagy pathway of T. cruzi is induced during metacyclogenesis. This work aimed to evaluate the participation of macroautophagy/autophagy in the distribution and function of reservosomes and cruzipain during this process. We found that parasite starvation promotes the cruzipain delivery to reservosomes. Enhanced autophagy increases acidity and hydrolytic activity in these compartments resulting in cruzipain enzymatic activation and self- processing. Inhibition of autophagy similarly impairs cruzipain traffic and activity than protease inhibitors, whereas mutant parasites that exhibit increased basal autophagy, also display increased cruzipain processing under control conditions. Further experiments showed that autophagy induced cruzipain activation and self-processing promote T. cruzi differentiation and host cell infection. These findings highlight the key role of T. cruzi autophagy in these processes and reveal a potential new target for Chagas disease therapy.

Abbreviations: Baf: bafilomycin A₁; CTE: C-terminal extension; Cz: cruzipain; IIF: indirect immunofluorescence; K777: vinyl sulfone with specific Cz inhibitory activity; Prot Inh: broad-spectrum protease inhibitor; Spa1: spautin-1; Wort: wortmannin

摘要

Cruzipain是致病性原生动物克氏锥虫的主要半胱氨酸蛋白酶，是一种重要的毒力因子，在寄生虫营养、分化和宿主细胞感染中起关键作用。Cruzipain 以酶原的形式合成、成熟并递送至储库体。这些通过内吞作用储存蛋白质和脂质的细胞器在T. cruzi 的后形成过程中数量急剧减少。自噬是在饥饿或不同压力情况下消化自身细胞成分以提供能量的过程。该途径在细胞生长、分化和死亡过程中很重要。之前，我们展示了T. cruzi的自噬途径在元环发生过程中被诱导。这项工作旨在评估巨自噬/自噬在此过程中参与储库体和cruzipain的分布和功能。我们发现寄生虫饥饿促进了cruzipain递送到储备体。增强的自噬会增加这些隔室中的酸度和水解活性，从而导致 cruzipain 酶促激活和自我加工。与蛋白酶抑制剂相比，抑制自噬同样会损害 cruzipain 的运输和活性，而表现出基础自噬增加的突变寄生虫在控制条件下也表现出增加的 cruzipain 加工。进一步的实验表明，自噬诱导的 cruzipain 激活和自我加工促进了T. cruzi分化和宿主细胞感染。这些发现强调了克氏锥虫自噬在这些过程中的关键作用，并揭示了南美锥虫病治疗的潜在新靶点。

缩写： Baf：巴弗洛霉素 A ₁；CTE：C端扩展；Cz：cruzipain；IIF：间接免疫荧光；K777：具有特定Cz抑制活性的乙烯基砜；Prot Inh：广谱蛋白酶抑制剂；Spa1：spautin-1；麦芽汁：渥曼青霉素