1 Universidade Federal de Santa Catarina, Brazil
DiscussionThe proliferation of the non-virulent protozoan Trypanosoma rangeli within its mammal hosts is controversial. Although increasing number of parasites are observed in experimental infections, there are no clues on the parasite replication sites and, thus, little is known how this species deal with the host’s defences. Assessment of cell cycle-associated proteins in T. rangeli revealed that the Polo-like kinase mRNA is up-regulated in proliferating epimastigotes, being a marker for cytokinesis. Although showing distinct expression levels and activity among T. rangeli strains from distinct genetic lineages, expression and enzymatic activity of the T. rangeli arginine kinase (TrAK) is down-regulated in blood trypomastigotes and up-regulated in epimastigotes, which may indicate a low or even absent replication of the bloodstream forms. These results allowed us to estimate that T. rangeli in vitro cell cycle takes ± 20.6 hours. The in vitro differentiation to infective trypomastigotes was assessed using distinct techniques. Along the classical morphological changes of the nucleus, kinetoplast and flagellum, increased mRNA levels of KMP-11, spermidine synthase (SpdS) and histidine ammonia lyase (HAL) were observed in epimastigotes while dihydrolipoamide dehydrogenase (DHLADH), mitochondrial HSP70 (mtHSP70), mitochondrial RNA binding protein 2 (MRP2), SS and a protein of unknown function revealed to be differentially expressed at the protein level between life-cycle stages during differentiation. A comparative in silico analysis of the genes related to the parasite antioxidant defence mechanisms, have shown that T. rangeli genome lacks the genes coding for cysthatione synthase (CS), ornithine decarboxylase (ODC), glutamylspermidine synthase (GspS), ascorbate peroxidase (AP) and one out of the three isoforms (“C”) of glutathione peroxidase (GP). However, multiple copies of cystathionine β-synthase (CβS) and GP isoform “A” were observed. Functional rescue of the CS activity in T. rangeli via heterologous expression of the L. amazonensis CS have doubled the epimastigotes survival rates when exposed to both oxidative (H2O2) and nitrosative (SNAP) stress if compared to wild-type parasites. Likewise, overexpression of a homologous trypanothione reductase (TR), which is present as a single copy gene on the haploid genome and shows reduced expression level in trypomastigotes, has also increased the parasite resistance to oxidative stress. We have also noticed that endogenous H2O2 production is higher in T. rangeli than T. cruzi epimastigotes. Taken together, our results seem to indicate that evolution of the T. rangeli antioxidant defence mechanisms is highly influenced by specific adaptations to the distinct biological life cycle within the insect vectors.
Funding: CNPq, CAPES, FINEP and STINT