Solid-phase binding assay of PMT to clusters 2, 3, and 4 of LRP1.

Graph depicts the binding curves of cluster 2 (dots), 3 (squares), and 4 (triangles) to plates coated with His-PMT. The equilibrium-binding affinity (KD) is presented inside the graph. Data are shown as mean with STDEV.</p

Oligonucelotides used in this study.

Oligonucelotides used in this study.</p

Antibodies used in this study.

Antibodies used in this study.</p

Expression of LRP1 or LRP1 cluster 4 in MEF-LRP1^-/- cells restores the ability of PMT to intoxicate cells.

MEF-LRP1-/- cells were transduced with retroviruses encoding LRP1 (top) or LRP1-cluster 4 (bottom), incubated in the presence of 5 nM PMT for the indicated time intervals, washed and lysed. Lysates were analyzed for toxin-induced modification of GαQ, for expression of LRP1 and for tubulin as loading control.</p

Table depicting the 10 mostly enriched sgRNAs and the function of the targeted genes.

(PDF)</p

Schematic presentation of the CRISPR/Cas9-Knock out screen.

Mouse embryonic fibroblasts (MEF) stably expressing Flag-Cas9-EGFP were transduced with a lentiviral CRISPR-library. Cells were treated three times with PMT(C1165S)DTa and surviving cells grown. Genomic DNA was extracted, inserted sgRNA amplified and sequenced.</p

Volcano plot showing significantly enriched genes.

The volcano plot shows gene expression changes between cells surviving treatment with usually lethal PMT-DTa chimera and those only transduced with the CRISPR library. All reads were mapped locally using BWA-MEM [5,6], then quantified with featureCounts [4], and finally fold changes between the condition were calculated by DESeq2 [7] (see galaxy history). The volcano plot was drawn with the bioinfokit toolkit [8]. Significantly enriched genes, having a positive fold change above 0.584 and a p-value lower or equal 5%, are shown in blue. The significance thresholds are marked by gray-dotted lines. The ten most significant genes are highlighted with their name. Non-significant genes are colored in gray.</p

LRP1 knockout or competitive inhibition of LRP1 inhibit modification of PMT.

MEF and MEF-LRP1-/- cells were incubated in the presence of 5nM PMT for the indicated time intervals, washed and lysed. Lysates were analyzed for toxin-induced modification of Gαq by an antibody detecting GαQ209E, for expression of LRP1 and for tubulin as loading control (top). MEF and MEF-LRP1-/- cells were incubated in the presence of 1 or 10 nM PMT in the presence or absence of GST or GST-RAP (1 μM), respectively, washed and lysed. Lysates were analyzed for toxin-induced modification of Gαq, for expression of LRP1 and for tubulin as loading control (c, bottom). Statistics: *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Expression levels of LRP1 do not exclusively determine the efficiency of intoxication.

HeLa and HepG2 cells were treated with the indicated concentrations of PMT for 2 h, or with 100 nM PMT for 16h as indicated, washed and lysed. A: Lysates were analyzed for toxin-induced modification of GαQ, LRP1 and GAPDH by Western-blotting. Shown is an example of 4 independent experiments. B: Quantification of the amount of LRP1 normalized to GAPDH in HeLa and HepG1 cells. C: HeLa and HepG2 cells were incubated with 1 μM Alexa 488 labeled PMT and washed. Cell bound fluorescence was analyzed by FACS in three independent experiments. Autofluorescence and relative cell size were used for normalizing bound fluorescence. Statistics: n.s: not significant, *, p (PDF)</p

Schematic presentation of the toxins used.

A: The Pasteurella multocida toxin (PMT) is composed of 5 domains: R: receptor binding domain, T: translocation domain containing two hydrophobic helices which are necessary for insertion into the endosomal membrane, C1-3: catalytic domain. C3 encodes for the deamidase domain. Mutation of C1165 to serine leads to a catalytically inactive toxin. B: Diphtheria toxin (DT) is composed of three domains: DTa is the catalytic domain, T: translocation domain, R: receptor binding domain. C: The fusion protein PMT(C1165S)DTa is composed of the catalytic inactive mutant of PMT and a c-terminally added catalytic domain of diphtheria toxin (DTa). (PDF)</p