CRISPR-Cas9 mutagenesis of U937 cells for variants resistant to Yersinia pestis intoxication

The plague agent, Yersinia pestis, employs a type III secretion system (T3SS) to selectively destroy human immune cells, thereby enabling its replication in the bloodstream and transmission to new hosts via fleabite. The host factors responsible for the selective destruction of immune cells by plague bacteria were not known. Here we show that LcrV, the needle cap protein of the Y. pestis T3SS, binds N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Overall design: Y. pestis AM18, a DyfeAB variant of the vaccine strain EV76, is defective for iron and manganese scavenging. In broth cultures, Y. pestis AM18 secretes the T3SS effector named YopE via the lcrV-dependent T3SS pathway. Compared with the DlcrV variant (AM46), which cannot kill immune cells above control levels, Y. pestis AM18 infection resulted in modest killing of U937 human histiocytic leukemia cells differentiated into macrophages (U937 cells). Y. pestis POO1 is a variant of AM18 expressing yopE-dtx, a translational hybrid between the YopE effector and the mature domain of diphtheria toxin. Dtx is an ADP-ribosyltransferase that modifies elongation factor 2 (EF2) at its diphthamide residue to promote cell death. Y. pestis POO1 secretes YopE-Dtx and causes death of U937 macrophages in an lcrV-dependent manner. U937 cells were transduced with lentiviral libraries expressing RNA guided endonuclease Cas9 and short guide RNAs (sgRNA – 3 per gene) targeting 19,052 genes. After 12 hours of infection with Y. pestis POO1 or POO2 (yopE-dtx ?lcrV), resistant cells were cultured with antibiotics to approximately 70% confluence and re-infected. The sgRNA sequences from surviving U937 cells were identified via next-generation sequencing (NGS) and candidate genes ranked based on the number of unique sgRNAs versus NGS reads. FPR1 stood out in three independent screens with the most abundant sgRNAs. FPR1 is a member of the GPCR family that activates immune cell chemotaxis and cytokine release in response to N-formylpeptides contained in bacteria or mitochondria.