The Trypanosoma cruzi vitamin C dependent peroxidase confers protection against oxidative stress but is not a determinant of virulence

Authors:
Martin C. Taylor, Michael D. Lewis, Amanda Fortes Francisco, Shane R. Wilkinson, John M. Kelly
In:
Source: Other
Publication Date: (2015)
Issue: 9(4): e0003707
Research Area:
Parasitology
Basic Research
Cells used in publication:
Trypanosoma cruzi
Species: unicellular
Tissue Origin:
Platform:
Nucleofector™ I/II/2b
Experiment
Trypanosoma cruzi epimastigotes (strain Sylvio X10/6) parasite transfection was carried out using a Nucleofector II device with human T-cell buffer (Lonza). 5E+7 epimastigotes were transformed with 5–10 µg of construct DNA. Drug selection was carried out at 10 µg ml-1 blasticidin, 5 µg ml-1 puromycin, 100 µg ml-1 G418 and 150 µg ml-1 hygromycin, as appropriate. Parasite cloning was carried out by diluting the parasite suspension to a concentration of 2 cells ml-1 and plating in 96-well microtitre plates at 100 µl per well. Plates were maintained at 27°C in 5% (v/v) CO2 with humidity. Lonza summary: The authors knocked-down successively both alleles of TcAPx gene in Trypanosoma cruzi using the Nucleofector II. They proved that TcAPx is not essential for parasite viability into its host cells, and do not play an important role chronic infections. They conclude that TcAPx should not be considered as a molecular target for future drug design against Chagas disease.
Abstract
BACKGROUND: The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy. METHODOLOGY/PRINCIPAL FINDINGS: To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo. CONCLUSIONS/SIGNIFICANCE: TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design.