Localization and Developmental Regulation of a Dispersed Gene Family 1 Protein in Trypanosoma cruzi

Abstract

The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the Trypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.Trypanosoma cruzi is the causative agent of Chagas disease, an endemic illness affecting between 16 and 18 million people in North, Central, and South America for which no vaccine or satisfactory treatment is available (22). During its life cycle, the parasite goes through different stages in the vector (epimastigotes and metacyclic trypomastigotes) and in the mammalian host (amastigotes and bloodstream trypomastigotes). As part of its survival strategy in these varying environments, the parasite has developed a large repertoire of multigene families (9,11,12,16). Among these families, the dispersed gene family 1 (DGF-1) has approximately 565 copies, ranging from 6 to 10 kbp, dispersed throughout the parasite genome (11). The members of the DGF-1 family encode proteins that share 85 to 95% sequence identity (11). Wincker and colleagues first identified clones bearing a common repeated sequence from a T. cruzi genome library (24) and later described the nucleotide sequence of a representative gene (DGF-1.1) (23). They concluded, from in silico studies, that DGF-1 genes encoded putative cell surface proteins (23). In 2005, Kim and colleagues (16) described a new member of this family (DGF-1.2) located in the subtelomeric region of a T. cruzi chromosome surrounded by mainly two kinds of sequences: genes encoding the trans-sialidase (TS) and retrotransposon hot spot (RHS) protein families. The sizes of the open reading frames (ORFs) of DGF-1 ge...

CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor

“…Western Blot Analysis-Western blotting analyses were performed using standard procedures used in our laboratory (42,43). Parental and mutant cell lines were harvested separately.…”

Hyperosmotic Stress Induces Aquaporin-dependent Cell Shrinkage, Polyphosphate Synthesis, Amino Acid Accumulation, and Global Gene Expression Changes in Trypanosoma cruzi

“…7A, calcium-binding protein and flagellar calcium-binding protein are down-regulated after 3 h of hyperosmotic stress, but they gradually recovered after continued incubation. The results of the Northern blot analyses of HSP10 and flagellar calcium-binding protein after 3,6, and 20 h of hyperosmotic stress were quantified and plotted together with the microarray results and are shown in Fig. 7, B and C, respectively.…”

Identification of genes encoding hypothetical proteins in open-reading frame expressed sequence tags from mammalian stages of Trypanosoma cruzi

“…DGF-1 proteins are encoded by ~500 genes (El-Sayed et al, 2005) and are localized in the parasite cell membrane. One of the DGF-1 members (DGF1.2) is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, and its expression is developmentally regulated (Lander et al, 2010). Interestingly, transcripts from DGF-1 have not been previously reported in T. cruzi EST databases (Table 2).…”