ty1 antibody  (DIAGENODE DIAGNOSTICS)

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: C-terminally tagged PfAlba2-Ty1, PfAlba3-Ty1, and PfAlba4-Ty1 were successfully expressed from an episome in P. falciparum asexual blood stages. ( A ) Schematic representation of the domain organization of the six Alba proteins of P. falciparum . ( B ) 3D7 parasites transfected with the pLN-PfAlba2-Ty1, pLN-PfAlba3-Ty1, or pLN-PfAlba4-Ty1 plasmids were grown in the presence of 5 mg/mL of blasticidin-S (BS), their genomic DNA harvested, and used with the indicated primers in PCRs to confirm the uptake of the episome. Primer pairs targeted either the genomic Alba locus ( p1 +p2 ) or different regions of the episome ( p3 +p4 and p5 +p6 ) as shown in the scheme ( top panel ), which is not drawn to scale. The DNA size marker corresponds to the GeneRuler 1 kb Plus DNA Ladder (Fermentas, Thermo Fisher Scientific). ( C ) PfAlba2-Ty1, PfAlba3-Ty1, or PfAlba4-Ty1 proteins were detected in protein lysates prepared from the indicated transfectant lines by Western blotting with mouse anti-Ty1 antibodies. PfHsp70 served as a loading control. ( D ) Immunofluorescence assays (IFAs) were used to determine the localization of PfAlba2-Ty1, PfAlba3-Ty1, and PfAlba4-Ty1 in ring (R), trophozoite (T), and schizont (S) stages of the indicated transfectant lines. Antibodies used included rabbit anti-Ty1 (red). Nuclei were labeled with DAPI (blue). Scale bar represents 5 µM. All of the cultures used for Western blotting and IFAs contained 5 µg/mL of blasticidin-S.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Transfection, Marker, Western Blot, Control, Immunofluorescence, Labeling

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: Overexpression of PfAlba2 or PfAlba3 adversely affects intra-erythrocytic growth of P. falciparum . ( A ) The growth of 3D7 + empty vector, 3D7 + PfAlba2-Ty1, 3D7 + PfAlba3-Ty1, and 3D7 + PfAlba4-Ty1 parasites was measured by flow cytometry for 5 days in the presence of the indicated concentrations of BS (in μg/mL). The y-axis denotes the percentage parasitemia at each time point. Data represent the means of a minimum of three independent experiments ± SEM (error bars). Red arrows indicate growth profiles of the strains at 10 µg/mL BS concentration. ( B ) The ~48-h IED cycle of the Alba-overexpressing lines was monitored by flow cytometry. The y-axis denotes the percentage of ring or schizont stages at each time point. Data represent the means of a minimum of three independent experiments ± SEM (error bars).

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Over Expression, Plasmid Preparation, Flow Cytometry, Concentration Assay

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: Transcriptomic data quality assessment by sample clustering demarcates the 3D7 + PfAlba2-ty1 and 3D7 + PfAlba3-Ty1 data sets from controls. ( A ) Schematic representation of the transcriptomics experiment. 3D7 + empty vector, 3D7 + PfAlba2-Ty1, 3D7 + PfAlba3-Ty1, or 3D7 + PfAlba4-Ty1 parasites were grown in white blood cell ( WBC )-free blood to ring (8–10 hpi) or trophozoite (28–30 hpi) stages in the presence of 5 µg/mL of blasticidin-S, total RNA harvested, and mRNA enriched and analyzed by strand-specific RNA-seq. Differential gene expression in the Alba-overexpressing lines relative to 3D7 and empty vector transfectants was quantified by DESeq2 analysis. ( B ) Principal component analysis of normalized read counts of 3D7, 3D7 + empty vector, 3D7 + PfAlba2-Ty1, 3D7 + PfAlba3-Ty1, and 3D7 + PfAlba4-Ty1 ring and trophozoite stage trancriptomes. Meaningful clustering of samples is indicated by dashed lines. ( C ) Pearson correlation coefficient R analysis of the normalized read counts from RNA-seq analysis of ring (R) and trophozoite (T) stage samples of the 3D7, 3D7 + empty vector, 3D7 + PfAlba2-Ty1, 3D7 + PfAlba3-Ty1, and 3D7 + PfAlba4-Ty1 strains. The color scale indicates the value of the R from 0 to 1. ( D ) Hours post-infection estimates for the transcriptomic data were obtained by passing the normalized RNA-seq data through the maximum likelihood algorithm developed by Lemieux et al . . The left panel shows the HPI estimates within 95% confidence intervals, while the right panel displays the actual likelihoods determined for each sample over the 48-h IED cycle.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Plasmid Preparation, RNA Sequencing, Gene Expression, Infection

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: PfAlba2 and PfAlba3 overexpression causes significant perturbations to the blood stage transcriptome. ( A ) Bar graph showing the total number of differentially expressed genes (DEGs) that are up- or downregulated in the indicated transgenic P. falciparum strain relative to controls. ( B ) Venn diagrams were used to represent the overlap in DEGs between the various Alba-overexpressing strains during ring and trophozoite stages. ( C ) Venn diagrams were used to represent the overlap in DEGs between ring and trophozoite stages of either 3D7 + PfAlba2-Ty1 or 3D7 + PfAlba3-Ty1 parasites. Troph = Trophozoite.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Over Expression, Transgenic Assay

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: Mistiming of key cellular developmental processes is linked to PfAlba3 overexpression. (A & B) Volcano plot of differentially expressed genes in ( A ) ring and ( B ) trophozoite stages of 3D7 + PfAlba3-Ty1 as compared to controls. Genes with |log 2 FC| > 1.5 and FDR < 0.05 values were considered to be differentially expressed (green dots). Pink dots indicate genes with |log 2 FC| > 1.5 but FDR > 0.05, purple dots indicate genes with |log 2 FC| < 1.5 but FDR < 0.05, and gray dots denote genes that are not changed significantly upon PfAlba3 overexpression. FC = fold change; FDR = false discovery rate. (C & D) Gene Ontology (GO) enrichment analysis in three categories, Cellular Component, Molecular Function, and Biological Process, for significantly up and downregulated genes in the ( C ) ring and ( D ) trophozoite stages of 3D7 + PfAlba3-Ty1 parasites. GO enrichment was performed after removing genes belonging to multigene families such as var and rifin . The number of genes enriched for each GO term relative to background is indicated on the right side of the y-axis , while the -log 10 (padj) of each GO term is represented on the left side of the y-axis . Note that p-adj is the same as FDR.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: Over Expression

Journal: Microbiology Spectrum

Article Title: Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

doi: 10.1128/spectrum.00885-24

Figure Lengend Snippet: Global var transcriptional repression is apparent in the PfAlba2- and PfAlba3-overexpressing parasites. ( A ) var gene transcriptional profile in ring stages was assessed by RNA-seq of P. falciparum 3D7 transfected with either PfAlba2-Ty1, PfAlba3-Ty1, or PfAlba4-Ty1 expression plasmids. Empty vector (pLN-Ty1) transfectants served as a control. The steady-state mRNA levels of all 60 var genes is indicated in reads per kilobase of transcript per million or RPKM ( y-axis ). Two replicates for each sample were analyzed. ( B ) Total mRNA levels (in RPKM) of the var gene family was calculated for two ring-stage RNA-seq replicates of each indicated strain. Data represent the mean + STDEV.

Article Snippet: Western blotting was performed with rabbit anti-Ty1 antibodies (Genscript), mouse BB2 monoclonal anti-Ty1 antibodies , rabbit anti-PfAlba2 antisera , mouse anti-PfAlba3 antisera , and rabbit anti-PfAlba4 antisera ( ).

Techniques: RNA Sequencing, Transfection, Expressing, Plasmid Preparation, Control