Importantly, in L. major and L. infantum, in which members of all four sub-families are found, amastin genes showed differences in genomic R428 positions and expression patterns of their mRNAs [8, 9]. More than fifteen years after their discovery, the function of amastins remains unknown. Because of the predicted structure and surface localization in the intracellular stage of T. cruzi and Leishmania spp, it has been proposed that amastins may play a role in host-parasite interactions within the mammalian cell: they could be involved in transport of ions, nutrients, across the membrane, or involved with cell signaling events that trigger parasite differentiation [9]. Its

preferential expression in the intracellular stage also suggest that it may constitute a relevant antigen during parasite infection, a prediction that was confirmed by studies showing that amastins peptides elicit strong immune response during Leishmanial infection [11]. Amastin antigens are considered a buy Adriamycin relevant immune biomarker of cutaneous and visceral Leishmaniasis as well as protective antigens in mice [12].

Although complete genome sequences of two strains of T. cruzi (CL Brener and SylvioX-10) have been reported, their assemblies were only partially achieved because of their unusually high repeat content [13, 14]. Therefore, for several multi-gene families, such as the amastin gene

family, their exact number of copies is not yet known. According to the current assembly [15], only four δ-amastins and two β-amastins were identified in the CL Brener genome. Herein, we used the entire data set of sequencing reads from the CL Brener [13] and Sylvio X-10 [14] genomes, to analyzed all sequences encoding amastin orthologues present in the HCS assay genomes of these two T. cruzi strains and determine their copy number as well as their genome organization. Expression of distinct amastin genes in fusion with the green fluorescent protein, Tolmetin allowed us to examine the cellular localization of different members of both amastin sub-families. By determining the levels of transcripts corresponding to each sub-family in all three parasite stages of various strains we showed that, whereas the levels of δ-amastins are up-regulated in amastigotes, β-amastin transcripts are significantly increased in the epimastigote insect stage. Most importantly, evidence indicating that amastins may constitute T. cruzi virulence factors was suggested by the analyses showing reduced expression of δ-amastins in amastigotes from strains known to have lower infection capacity. Results and discussion The amastin gene repertoire of Trypanosoma cruzi In its current assembly, the T. cruzi (CL Brener) genome exhibits 12 putative amastin sequences.