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Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens
Published on: June 6, 2020
Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein
Julian Schoellkopf1, Thomas Mueller1, Lena Hippchen1
1Institute for Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, Albert-Ludwigs-University, Freiburg, Germany.
Abstract:
PMT is a protein toxin produced by Pasteurella multocida serotypes A and D. As causative agent of atrophic rhinitis in swine, it leads to rapid degradation of the nasal turbinate bone. The toxin acts as a deamidase to modify a crucial glutamine in heterotrimeric G proteins, which results in constitutive activation of the G proteins and permanent stimulation of numerous downstream signaling pathways. Using a lentiviral based genome wide CRISPR knockout screen in combination with a lethal toxin chimera, consisting of full length inactive PMT and the catalytic domain of diphtheria toxin, we identified the LRP1 gene encoding the Low-Density Lipoprotein Receptor-related protein 1 as a critical host factor for PMT function. Loss of LRP1 reduced PMT binding and abolished the cellular response and deamidation of heterotrimeric G proteins, confirming LRP1 to be crucial for PMT uptake. Expression of LRP1 or cluster 4 of LRP1 restored intoxication of the knockout cells. In summary our data demonstrate LRP1 as crucial host entry factor for PMT intoxication by acting as its primary cell surface receptor.
Insights
Pasteurella multocida toxin (PMT) causes swine atrophic rhinitis by degrading nasal bone. Researchers found Low-Density Lipoprotein Receptor-related protein 1 (LRP1) is essential for PMT entry into cells.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Pasteurella multocida toxin (PMT) is a deamidase causing swine atrophic rhinitis.
- PMT constitutively activates heterotrimeric G proteins, leading to signaling pathway stimulation.
- The host factors mediating PMT cell entry and intoxication remain largely unknown.
Purpose of the Study:
- To identify host factors critical for PMT cell entry and intoxication.
- To elucidate the mechanism by which PMT interacts with host cells.
Main Methods:
- Genome-wide CRISPR knockout screen using a lentiviral delivery system.
- Utilized a lethal toxin chimera (inactive PMT fused to diphtheria toxin catalytic domain).
- Assessed PMT binding, cellular response, and G protein deamidation in knockout cells.
Main Results:
- Identified the Low-Density Lipoprotein Receptor-related protein 1 (LRP1) gene as essential for PMT function.
- Loss of LRP1 significantly reduced PMT binding and abolished cellular intoxication.
- Re-expression of LRP1 restored PMT uptake and deamidation activity in knockout cells.
Conclusions:
- LRP1 acts as the primary cell surface receptor for PMT.
- LRP1 is a crucial host entry factor for PMT intoxication in susceptible cells.
- Understanding LRP1's role provides insights into PMT pathogenesis and potential therapeutic targets.
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