fasciculata. In addition, two kinetoplast-associated proteins of T. cruzi, TcKAP4 and TcKAP6, were cloned, expressed and antisera were generated against recombinant proteins. Imunolabeling

assays revealed a differential distribution of TcKAPs in the kinetoplast of distinct developmental stages of the parasite. Methods Cell culture Epimastigote forms of T. cruzi (Dm28c clone) [22] MDV3100 mw were grown in liver infusion tryptose (LIT) medium supplemented with 10% fetal calf serum at 28°C. Bloodstream trypomastigote forms derived from the blood of Swiss mice were used to infect the LLC-MK2 cells. Trypomastigotes were released seven days after infection in the supernatant and purified by centrifugation. Amastigotes were obtained by Selleckchem ZD1839 disruption of the LLC-MK2 cells after four days of infection with trypomastigotes. It is worth mentioning that the amastigotes released after disruption of the cells

are mixed with intermediate forms, which PR-171 mw represent a transitional stage between amastigotes and trypomastigotes [20]. DNA extraction DNA was extracted as described by Medina-Acosta and Cross [23]. Genome search for T. cruzi orthologs of CfKAPs The CfKAPs1–4 protein sequences were retrieved from GenBank® [24] and a BLASTp search [25] was performed against all protein sequences

from trypanosomatids with a complete sequenced genome, available in GenBank® (release 169). All hits having an e-value lower than 1e10-5 were selected for further analyses. Sequences that were redundant or did not contain a discernible nine amino acids presequence, suggestive of kinetoplast import, were discarded. Evolutionary P-type ATPase analysis of trypanosomatids KAPs Multiple sequence alignments (MSAs) were produced with the ClustalW software [26] and a phylogenetic analysis was performed using the MrBayes software [27, 28], running in parallel [29] in a 28 nodes cluster, by 20,000,000 generations, with gamma correction (estimated α = 6.675), allowing for invariant sites. A mixed amino acid model was used and the Wag fixed rate model [30] prevailed with a posterior probability of 1.0. MSAs and trees were visualized with the Jalview [31] and TreeView software [32], respectively Cloning and expression of the TcKAP4 and TcKAP6 genes Primers were designed to amplify the entire coding region of these genes from the T. cruzi Dm28c genome.