Infection by Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, depends on reactive oxygen species (ROS), which has been described to induce parasite proliferation in mammalian host cells. It is unknown how the parasite manages to increase host ROS levels. Here, we found that intracellular T. cruzi forms release in the host cytosol its major cyclophilin of 19 kDa (TcCyp19). Parasites depleted of TcCyp19 by using CRISPR/Cas9 gene replacement proliferate inefficiently and fail to increase ROS, compared to wild type parasites or parasites with restored TcCyp19 gene expression. Expression of TcCyp19 in L6 rat myoblast increased ROS levels and restored the proliferation of TcCyp19 depleted parasites. These events could also be inhibited by cyclosporin A, (a cyclophilin inhibitor), and by polyethylene glycol‐linked to antioxidant enzymes. TcCyp19 was found more concentrated in the membrane leading edges of the host cells in regions that also accumulate phosphorylated p47phox, as observed to the endogenous cyclophilin A, suggesting some mechanisms involved with the translocation process of the regulatory subunit p47phox in the activation of the NADPH oxidase enzymatic complex. We concluded that cyclophilin released in the host cell cytosol by T. cruzi mediates the increase of ROS, required to boost parasite proliferation in mammalian hosts.
The integrated stress response in eukaryotic cells is an orchestrated pathway that leads to eukaryotic Initiation Factor 2 alpha subunit (eIF2α) phosphorylation at ser51 and ultimately activates pathways to mitigate cellular damages. Three putative kinases (Tck1, Tck2, and Tck3) are found in the Trypanosoma cruzi genome, the flagellated parasite that causes Chagas disease. These kinases present similarities to other eukaryotic eIF2α kinases, exhibiting a typical insertion loop in the kinase domain of the protein. We found that this insertion loop is conserved among kinase 1 of several T. cruzi strains but differs among various Kinetoplastidae species, suggesting unique roles. Kinase 1 is orthologous of GCN2 of several eukaryotes, which have been implicated in the eIF2α ser51 phosphorylation in situations that mainly affects the nutrients levels. Therefore, we further investigated the responses to nutritional stress of T. cruzi devoid of TcK1 generated by CRISPR/Cas9 gene replacement. In nutrient-rich conditions, replicative T. cruzi epimastigotes depleted of TcK1 proliferate as wild type cells but showed increased levels of polysomes relative to monosomes. Upon nutritional deprivation, the polysomes decreased more than in TcK1 depleted line. However, eIF2α is still phosphorylated in TcK1 depleted line, as in wild type parasites. eIF2α phosphorylation increased at longer incubations times, but KO parasites showed less accumulation of ribonucleoprotein granules containing ATP-dependent RNA helicase involved in mRNA turnover (DHH1) and Poly-A binding protein (PABP1). Additionally, the formation of metacyclic-trypomastigotes is increased in the absence of Tck1 compared to controls. These metacyclics, as well as tissue culture trypomastigotes derived from the TcK1 knockout line, were less infective to mammalian host cells, although replicated faster inside mammalian cells. These results indicate that GCN2-like kinase in T. cruzi affects stress granule formation, independently of eIF2α phosphorylation upon nutrient deprivation. It also modulates the fate of the parasites during differentiation, invasion, and intracellular proliferation.
Human infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease for which there are no prophylactic vaccines. Cyclophilin 19 is a secreted cis-trans peptidyl isomerase expressed in all life stages of Trypanosoma cruzi. This protein in the insect stage leads to the inactivation of insect anti-parasitic peptides and parasite transformation whereas in the intracellular amastigotes it participates in generating ROS promoting the growth of parasites. We have generated a parasite mutant with depleted expression of Cyp19 by removal of 2 of 3 genes encoding this protein using double allelic homologous recombination. The mutant parasite line failed to replicate when inoculated into host cells in vitro or in mice indicating that Cyp19 is critical for infectivity. The mutant parasite line also fails to replicate in or cause clinical disease in immuno-deficient mice further validating their lack of virulence. Repeated inoculation of mutant parasites into immuno-competent mice elicits parasite-specific trypanolytic antibodies and a Th-1 biased immune response and challenge of mutant immunized mice with virulent wild-type parasites is 100% effective at preventing death from acute disease. These results suggest that parasite Cyp19 may be candidate for small molecule drug targeting and that the mutant parasite line may warrant further immunization studies for prevention of Chagas disease.
Ubiquitylation is a post-translational modification promoting protein degradation. Within the endomembrane system ubiquitylation marks proteins for lysosomal processing. Deubiquitinases (DUBs) cleave ubiquitin from modified proteins and, in the case of surface proteins, prevent lysosomal targeting and thus play a major role in controlling turnover. In Trypanosoma cruzi, the etiological agent of Chagas disease, acquisition of nutrients in parasites proliferating within the blood meal of insect vectors occurs via the cytostome, a unique structure connected to a long tubular cytopharynx. Contents are delivered to late endosomes and accumulate in reservosomes, equivalent to lysosomes. When starved, T. cruzi differentiates into mammalian infective trypomastigotes, which are cell cycle arrested. Here we asked what roles ubiquitylation plays in this unique endocytic process by interrogation of the T. cruzi ortholog of VDU (von Hippel-Lindau-interacting deubiquitylating enzyme)/USP33 (ubiquitin-specific protease). We found that TcVDU expression level inversely correlated with transferrin endocytosis, and that overexpression led to a longer retention of the endocytic cargo near the cytostome. TcVDU itself was found enriched in the anterior region of the parasite, in proximity to endocytic cargo. Most importantly, TcVDU overexpression reduced parasite invasion capacity and led to increased release of trans-sialidase. These alterations in the abundance of multiple surface proteins in TcVDU mutants indicate a key role of TcVDU in modulating the T. cruzi surface by affecting the endosomal traffic and consequently the host-parasite interface.
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