Antibodies used in this study.

Antibodies used in this study.</p

Oligonucelotides used in this study.

Oligonucelotides 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

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

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

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

LRP1 knockout prohibits binding and uptake of PMT.

MEF (circles) and MEF-LRP1-/- cells (squares), respectively were incubated with increasing concentrations of PMT(C1165S)DTa for 48h and cell viability was analyzed as % of untreated controls. Shown is the mean of three independent experiments (A). Cells were incubated with increasing concentrations of Alexa488 labeled PMT (PMT488), washed and bound fluorescence analyzed by FACS shown as mean of three independent experiments (B).</p