Mice immunization was performed administering four 100 g doses of rTcMVK at 15 day intervals
Mice immunization was performed administering four 100 g doses of rTcMVK at 15 day intervals. isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for invasion. is the etiological agent of Chagas disease or American trypanosomiasis, a prevalent health problem that affects 6C7 million people worldwide, mostly in Latin America1. has a complex life cycle including invertebrate and vertebrate hosts. Trypomastigotes and extracellular amastigotes are the infective forms for mammalian hosts and they may participate a variety of strategies to PETCM infect and survive in mammalian host cells2,3. does not synthesize cholesterol and sterol biosynthesis is usually distributed in multiple intracellular compartments and the production of HMG-CoA from acetyl Coenzyme A and generation of mevalonate mainly occurs in the mitochondrion while further mevalonate phosphorylation is almost exclusively located in glycosomes8. During the course of the characterization of MVK (TcMVK), we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion, possibly by phosphorylation of host cell components. Given this unexpected behavior we hypothesized that TcMVK may be a bifunctional enzyme, typified by having a second function not related to ITGA2B its initial, classical function. In addition, many enzymes that are originally involved in metabolic pathways can also act as PETCM virulence factors of pathogenic protozoa9,10,11,12. The present study demonstrates a new role for any metabolic enzyme of isolates used in this study were: strain CL and clone CL Brener (DTU VI13,14), and strain G (DTU I13,15). Extracellular amastigotes (EAs) were obtained by differentiation of tissue culture trypomastigotes (TCTs) in LIT medium at pH 5.8 for 14?h as previously described16. Epimastigotes (EPs) and metacyclic trypomastigotes (MTs) were obtained as previously explained17. HeLa cells (Instituto Adolfo Lutz, S?o Paulo, SP, Brazil) were grown in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 10?g/mL streptomycin, 100?U/mL penicillin and 40?g/mL gentamycin at 37?C and 5% CO2. Invasion assays HeLa cell invasion assays were performed by adding 500?L of cell suspension (1.5??105) to 24 well plates containing sterile glass coverslips and incubated overnight at 37?C and 5% CO2. Cells were treated with 300?nM of rTcMVK in RPMI/10% FBS and incubated at 37?C in a CO2 (5%) humidified incubator for 1?h and washed twice with sterile PBS. Next, parasite suspensions at 10:1 per cell for EAs and 20:1 for MTs were added and the plates incubated for another 2?h at 37?C in a 5% CO2 humidified incubator. The cells were then softly washed three times with PBS to remove unattached parasites, fixed with Bouin and stained with Giemsa as previously explained18. For antibody inhibition assays parasites were incubated for 30?min with anti-MVK, specificity control -C0319, rTcMVK PETCM or untreated, washed then incubated with HeLa cells as described above. Cloning of PETCM TcMVK alleles Alleles were amplified by PCR from genomic DNA of clone CL Brener, G and CL strains and cloned into pGEM?-T Easy Vector (Promega, Fitchburg, WI, USA). The coding sequences of TcMVK were amplified using the following primers: “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_797435.1″,”term_id”:”71397723″,”term_text”:”XM_797435.1″XM_797435.1 (Esmeraldo PETCM like), forward- 5 CTA AAT TTT GGC Take action TCT AGG GCA 3 and Reverse: GAA GTA CAG GAA CGT TAT TTA ACC T; and “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_809535.1″,”term_id”:”71651923″,”term_text”:”XM_809535.1″XM_809535.1 (non-Esmeraldo like), forward ultra II DNA polymerase (Agilent, Santa Clara, CA, USA) in a final volume of 50?L. PCR conditions were: 35 cycles of 1 1?min at 94?C, 1?min at 94?C, 30?s at 50?C, 1?min at 72?C, and a final extension of 10?min at 72?C. BL21 strain cells were transformed with pET-28a:TcMVK plasmid and produced at 20?C for 48?h, 150?rpm, in 4?L ZYM-5052 high induction medium21 containing kanamycin (30?g/mL). Cells were harvested by centrifugation (40?min, 6000?g), the pellet re-suspended in one tenth of culture volume in buffer A (50?mM Tris, 250?mM NaCl, 20?mM imidazole, pH 7.5) and stored at ?20?C. Purification of rTcMVK Cells were incubated with lysozyme (100?M) in an ice bath for 40 min and then were lysed by sonication using a Branson Sonifier 450 (VWR Scientific, Radnor PA, USA) equipped with a 2?mm-diameter tip. Debris was removed by.