Establishing the role of the mitochondrial voltage-dependent anion channel (VDAC) in Trypanosoma cruzi.

Abstract

The protozoan Trypanosoma cruzi is the causative agent of Chagas Disease, a neglected tropical disease that affects millions globally. As it transitions from insect vector to human host, T. cruzi faces homeostatic challenges, including oxidative stress, fluctuations in osmolarity, and nutrient availability. Ion channels regulate the parasite's ability to respond to environmental stressors. Our group has previously identified a mechanosensitive channel located in the contractile vacuole that, when knocked out, leads to reduced infectivity, abnormal morphology, and impaired motility. Transcriptomic analysis showed that Voltage-Dependent Anion Channel (VDAC) was significantly downregulated in the knockouts, suggesting a functional link between the two proteins. VDAC is a highly expressed outer mitochondrial membrane protein that, in other eukaryotes, regulates cell death, mitochondrial dynamics and metabolism. In protozoans, VDAC has been shown to share similar functions, with additional roles in tRNA and protein import. However, the function of VDAC in T. cruzi remains uncharacterized. The genome of T. cruzi contains five copies of VDAC sharing 99% homology. Four of them are organized in tandem, suggesting a gene expansion event as their most probable origin. To elucidate the function of this channel in the parasites, we targeted a conserved region using CRISPR/Cas9 strategies to generate a VDAC-deficient cell line. The mutant strain exhibited abnormal morphology and significant growth defects relative to the wild type, suggesting that VDAC is critical for parasite replication. Fluorometric analysis indicates that VDAC downregulation results in mitochondrial depolarization and reduced calcium uptake supporting its role in maintenance of mitochondrial functions.