generation of the consensus sequence  (STAB VIDA)

Journal: The Journal of Biological Chemistry

Article Title: Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

doi: 10.1016/j.jbc.2023.104857

Figure Lengend Snippet: Epimastigote and amastigote proliferation is inhibited by TcK2 depletion, while differentiation in metacyclics and trypomastigote invasion are preserved . A , proliferation of the parental (Cas9) and TcK2-depleted epimastigote parasites in normal medium. The values are means of triplicate experiments. B , late exponential epimastigote cultures (2 × 10 7 /ml) were washed and incubated in Grace’s medium. After 7 days, the total number of Epis, Intermediate Forms (IF), and Metas were estimated by Giemsa staining of 250 parasites in three independent experiments. C , L6 rat myoblasts (1 × 10 5 ) were plated on round coverslips in 24-well plates. After 24 h, the cells were incubated with 0.5 ml purified metacyclics at the indicated multiplicity of infection (MOIs) for 3 h at 37 °C, and the number of internalized parasites per cell was counted by Giemsa staining as described in Methods. D , ROS levels measured with CM-H2DCFDA of epimastigotes and ( E ) of L6 and L6 expressing T. cruzi cyclophilin 19. The values are triplicate experiments and with p = 0.05 and 0.14, respectively, calculated by two-tailed unpaired t test. F and G , egress of TCT from L6 myoblast, and L6 myoblast overexpressing T. cruzi cyclophilin 19, respectively, infected for 3 h to obtain the same infectivity with Cas9 and TcK2KO parasites TCTs. The graphs show the daily number of TCTs collected in the culture supernatant. All the values are means and SD (n = 3), and asterisks indicate p < 0.05 using two-way ANOVA.

Article Snippet: The first infection was made with metacyclic and the subsequent with the TCT released on day 7 post-infection. (D) Agarose gel electrophoresis of the PCR product of BSD resistant epimastigotes. (E) Comparison of the original sequence of the TcK2 ORF (Cas9) with the consensus alignment generated by Illumina sequence of the TcK2KO.

Techniques: Incubation, Staining, Purification, Infection, Expressing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

doi: 10.1016/j.jbc.2023.104857

Figure Lengend Snippet: Global proteome changes upon TcK2 knockout for three T. cruzi life-cycle stages . Volcano plots of −log 10 t test p -value plotted versus the t test difference (difference between means) comparing protein abundance of TcK2KO cells with the respective parental cells—for ( A ) intracellular amastigotes collected 72 h after infection (Amas), ( B ) exponentially growing epimastigotes (Epis), and ( C ) TCTs released from infected L6 cells. The complete results from label-free quantification are in Table S3 . The green symbols show increased and red decreased levels in TcK2KO relative to the parental line (Cas9). The annotated names are show with the respective accession number in https://tritrypdb.org . The blue dot indicates eIF3a.

Article Snippet: The first infection was made with metacyclic and the subsequent with the TCT released on day 7 post-infection. (D) Agarose gel electrophoresis of the PCR product of BSD resistant epimastigotes. (E) Comparison of the original sequence of the TcK2 ORF (Cas9) with the consensus alignment generated by Illumina sequence of the TcK2KO.

Techniques: Knock-Out, Infection

Journal: The Journal of Biological Chemistry

Article Title: Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

doi: 10.1016/j.jbc.2023.104857

Figure Lengend Snippet: Trans-sialidase expression is increased in intracellular amastigotes of TcK2KO. Western blot of the Cas9 and TcK2KO of epimastigote (Epi), metacyclic-trypomastigotes (Meta), intracellular amastigotes (Ama), and tissue culture–derived trypomastigotes (TCT) extracts, each containing 20 μg of protein, probed with trans-sialidase monoclonal antibody (mAb 39, green ) and anti HSP70. Lane M indicates the molecular weight markers of each gel.

Article Snippet: The first infection was made with metacyclic and the subsequent with the TCT released on day 7 post-infection. (D) Agarose gel electrophoresis of the PCR product of BSD resistant epimastigotes. (E) Comparison of the original sequence of the TcK2 ORF (Cas9) with the consensus alignment generated by Illumina sequence of the TcK2KO.

Techniques: Expressing, Western Blot, Derivative Assay, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

doi: 10.1016/j.jbc.2023.104857

Figure Lengend Snippet: Major protein phosphorylation changes between parental TcK2KO lines of T. cruzi stages . Volcano plots of −log 10 t test p -value plotted versus the t test difference (difference between means) comparing phosphoprotein abundance of TcK2KO cells with the respective parental cells for ( A ) intracellular amastigotes collected 72 h after infection (Amas), ( B ) exponentially growing epimastigotes (Epis), and ( C ) trypomastigotes released from infected L6 cells (see also Table S3 ). Green symbols are proteins increased and red decreased in the TcK2KO relative to the control samples. The corresponding proteins with the respective accession number in https://tritrypdb.org and with the respective peptides and phosphorylation sites are shown in the figure.

Article Snippet: The first infection was made with metacyclic and the subsequent with the TCT released on day 7 post-infection. (D) Agarose gel electrophoresis of the PCR product of BSD resistant epimastigotes. (E) Comparison of the original sequence of the TcK2 ORF (Cas9) with the consensus alignment generated by Illumina sequence of the TcK2KO.

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

doi: 10.1016/j.jbc.2023.104857

Figure Lengend Snippet: TcK2 specificity in vivo and in vitro . Western blot of extracts containing 1 × 10 7 parasites of parental (Cas9) and TcK2KO epimastigotes incubated in LIT or TAU solution for 2 h, at 28 °C and probed with rabbit anti-phospho-eIF2α ( top panel ), total mouse anti-eIF2α ( middle panel ), and rabbit anti-eIF2α developed with the respective IRDye anti-IgG conjugates and anti-HSP70. B , relative phosphorylation of triplicate experiments for the parental and TcK2KO lines incubated in TAU as in ( A ) compared with parasites maintained in LIT. The ratio was measured relative to total eIF2α or HSP70 as indicated. The asterisks indicate p < 0.05 calculated by two-way ANOVA. C , Western blots of samples containing the indicated concentrations of recombinant mouse PERK kinase domain or TcK2 (KD4) for 1 h, at 28 °C in the presence of 10 μM recombinant mouse eIF2α and 0.2 mM ATP. The blots were decorated with anti-phospho-Ser51 antibodies and anti-eIF2α. D , Western blots of samples of recombinant mouse eIF2α (10 μM) incubated with ATP (0.1 mM) and 100 nM of recombinant PERK or TcK2 (KD4) for the indicated times and decorated as above. E , effect of TcK2 concentrations on the phosphorylation of synthetic peptides used as substrate: CREBtide, histone H3, and p70S6K, by employing the LANCE assay after 3-h incubation.

Article Snippet: The first infection was made with metacyclic and the subsequent with the TCT released on day 7 post-infection. (D) Agarose gel electrophoresis of the PCR product of BSD resistant epimastigotes. (E) Comparison of the original sequence of the TcK2 ORF (Cas9) with the consensus alignment generated by Illumina sequence of the TcK2KO.

Techniques: In Vivo, In Vitro, Western Blot, Incubation, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

doi: 10.1016/j.jbc.2023.104857

Figure Lengend Snippet: TcK2KO proliferating amastigotes are more resistant to Dasatinib than wildtype parasites . L6 rat myoblasts (1 × 10 3 ) were seeded in black 96-well plates with transparent bottom in 120 μl of DMEM medium containing 10% FBS. After 24 h, 30 μl of TCT of the Cas9 (A) or TcK2KO line (first passage, B ) was added (MOI=20) and the cells were maintained at 37 °C. After the next 24 h, serial dilutions of Dasatinib were added in a volume of 30 μl and the incubation was continued for 72 h, before fixing the cells with 4% p -formaldehyde in PBS, washing once with PBS and staining with Draq5. Imaging was carried out using an automated cell plate reader. Infection ratio (IR) was defined as the ratio between (i) the total number of infected cells in all images from the well and (ii) the total number of cells in all images from the same well. The values are means and standard deviations from duplicate experiments, each one made in two wells for each concentration.

Article Snippet: The first infection was made with metacyclic and the subsequent with the TCT released on day 7 post-infection. (D) Agarose gel electrophoresis of the PCR product of BSD resistant epimastigotes. (E) Comparison of the original sequence of the TcK2 ORF (Cas9) with the consensus alignment generated by Illumina sequence of the TcK2KO.

Techniques: Incubation, Staining, Imaging, Infection, Concentration Assay